Pyronin antibacterials, process and novel intermediates thereto

ABSTRACT

The present invention provides convergent processes for preparing myxopyronins and corallopyronins, compounds useful as antibacterial therapeutics. The present invention also provides novel compositions of matter which are useful for the preparation of pyronin antibiotics.

This is a division of application Ser. No. 09/002,541, filed Jan. 2,1998, now U.S. Pat. No. 6,022,983, which is a continuation-in-part ofapplication Ser. No. 08/822,323, filed Mar. 21, 1997, now U.S. Pat. No.5,986,111, which claimed priority under 35 U.S.C. §119 of provisionalapplication Ser. No. 60/013,874, filed Mar. 22, 1996. Each of theseprior applications is hereby incorporated herein by reference, in itsentirety.

FIELD OF THE INVENTION

The present invention is in the field of pyronin antibiotics. Inparticular, the present invention relates to processes for thepreparation of myxopyronins and corallopyronins, compounds useful asantibacterial therapeutics. The present invention also provides novelcompositions of matter which are useful as intermediates for preparingthe pyronin antibiotics.

Throughout this application, various publications are referred to, eachof which is hereby incorporated by reference in its entirety into thisapplication to more fully describe the state of the art to which theinvention pertains.

BACKGROUND OF THE INVENTION

Myxopyronins and corallopyronins are 2-pyrone-containing antibioticswhich present a significant opportunity in antibacterial therapy. Theyconstitute a synthetically accessible, unexploited series of lowmolecular weight bacterial RNA polymerase (RNAP) inhibitors withfavorable properties: selectivity vs. human RNAP, cell penetration(minimal inhibitory concentrations (MICs) at concentrations comparableto in vitro bacterial RNAP IC₅₀s), and potency againstrifampicin-resistant S. aureus equal to that against arifampicin-susceptible strain.

Corallopyronin A/B and myxopyronin A/B are natural products isolatedfrom gliding bacteria (Corallococcus coralloides; Myxococcus fulvus) anddiscovered to be RNAP inhibitors. Reichenbach, H., et al., Liebigs Ann.Chem., 1983, 1656; Reichenbach, H., et al., Liebigs Ann. Chem., 1984,1088; Reichenbach, H., et al., Liebigs Ann. Chem., 1985, 822. Thestructures of these compounds are closely related having in common a3-acyl-4-hydroxy-2-pyrone with an alkyl chain at the 6-position bearinga vinyl carbamate functionality, a feature atypical of natural products.They differ only in the substitution on the alkyl chain attached to the3-position of the pyrone, the corallopyronins being more elaborate (FIG.1 (a)). The pyronins have good intrinsic activity in antibacterialassays against both E. coli and S. aureus RNA polymerase. This activityis specific with respect to human or SP6 polymerases. MIC data (seeTable I) show that these compounds, like rifampicin, are not absorbedwell by E. coli but that they have intrinsic activity against both grampositive and gram negative bacteria.

TABLE I In Vitro IC₅₀ (μM) MIC (μg/ml) E.coli S.aureus Human SP6 E. coliE. coli Compound RNAP RNAP RNAP RNAP Rev/RRE S.aureus MCR BASMyxopyronin A/B 10 10 >200 >200 100 4 180 1.6 Corallopyronin A/B 610 >200 >200 100 4 >200 0.4

An attractive feature of this series of compounds is their activityagainst strains resistant to rifampicin. The MIC for rifampicin is ca.10 nM against susceptible strains, but falls off against resistantstrains (MIC>10 μM). The use of rifampicin is limited by the developmentof bacterial resistance. Both myxo- and corallopyronins are equiactiveagainst Rif-susceptible and Rif-resistant S. aureus.

Pyrones have been used in the prior art to elicit a biological effect ina few instances, but in none of these instances have they been used asan antibacterial agent. 2H-Pyran-2,6(3H)-dione derivatives are reportedto be active at reasonable doses in a passive cutaneous anaphylaxismodel in rats when administered by either the intravenous or oral route.Snader, K. M. et al., J. Med. Chem., 1979, 22, 706; Chahrin, L. W.,Snader, K. M., Williams, C. R., 2H-Pyran-2,6(3H)-dionederivate. GermanPatent 25 33 843. In a second case, simple3-(1-oxoalkyl)-4-hydroxy-6-alkyl-2-pyrones were found to be effective invitro in the inhibition of human sputum elastase. Cook, L., Ternai, B.,Ghosh, P., J. Med. Chem., 1987, 30, 1017. Lastly, a series of pyronederivatives were found to be effective inhibitors of HIV protease inboth enzymatic assays and cell culture (FIG. 1(b)). Skulnick, H. I., etal., J. Med. Chem., 1995, 38, 4968. No synthetic investigations ormedicinal uses of pyronin antibacterials have been reported in theliterature.

The present invention provides novel intermediates useful in thesynthesis of myxopyronins A and B and derivatives thereof. In addition,the present invention provides processes for synthesizing myxopyronins Aand B and derivatives thereof as well as corallopyronins. Themyxopyronins of the invention are useful against gram negative andpositive bacteria.

SUMMARY OF THE INVENTION

One object of the present invention is to provide processes for thepreparation of myxopyronins and corallopyronins, compounds useful asantibacterial therapeutics. In particular, the present inventionprovides myxopyronins A and B.

Another object of the present invention is to provide variouscompositions of matter useful as intermediates in the preparation of theantibiotic myxopyronin.

A further object of the present invention is to provide methods ofpreparing such intermediates.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(a) shows the structures of corallopyronin A/B and myxopyroninA/B.

FIG. 1(b) illustrates coumarin derivatives which inhibit HIV protease inenzymatic assays and cell culture. Skulnick, H. I., et al., J. Med.Chem., 1995, 38, 4968.

FIG. 2 illustrates a retrosynthetic analysis for the preparation ofmyxopyronin A.

FIG. 3 illustrates the synthesis of compound 1 in accord with thepresent invention. (The natural products have been illustrated with thenatural occurring (R)-configuration, however all materials in this paperwere synthesized in racemic form.)

FIG. 4 illustrates alkylation of the 7-position of compound 3 for thepreparation of compound 10.

FIG. 5 illustrates the synthesis of compound 6 in accord with thepresent invention.

FIGS. 6a and 6 b Illustrate the synthesis of compound 10 in accord withthe present invention.

FIGS. 7a and 7 b Illustrate an alternative synthesis of compound 10 inaccord with the present invention.

FIGS. 8aand 8 b Illustrates the synthesis of compound 18 in accord withthe present invention.

FIG. 9 illustrates the attempted acylation of pyrone 10 with acylchloride 19.

FIGS. 10a and 10 b Illustrates an alternative retrosynthetic analysisfor the preparation of myxopyronin A.

FIGS. 11a and 11 b Illustrates the acylation of pyrone 10 with propionylchloride and subsequent elaboration to myxopyronin A.

FIG. 12 shows a retrosynthetic analysis of myxopyronin A.

FIG. 12(a) shows the preparation of allyl aldehyde 14.

FIG. 12(b) shows the preparation of acylated pyrone 5.

FIG. 12(c) shows the preparation of iodide 8 b.

FIG. 13(a) provides a synthetic route to acyl pyrone intermediate 15.

FIG. 13(b) illustrates the condensation reaction of intermediates 14 and15 and subsequent elaboration to myxopyronin A.

FIG. 14 shows the preparation of allyl aldehyde 14.

FIG. 15 provides a synthetic route to intermediate pyrone 10.

FIG. 16 illustrates a retrosynthetic analysis of myxopyronin A and B.

FIG. 17 provides a synthetic route to compound 15 a.

FIG. 18 shows a method for preparing α,β-unsaturated aldehydes useful asintermediates in the synthesis of myxopyronins.

FIG. 19 shows a synthetic route to myxopyronin A and B starting fromintermediate 15 a.

FIG. 20(a) provides a graphical representation of the inhibitory effectas a function of concentration of a mixture of isolated naturalmyxopyronins A and B, compared with synthetic racemic myxopyronin A andrifampicin, in an in vitro transcription assay using E. coli RNApolymerase.

FIG. 20(b) provides a graphical representation of the inhibitory effectas a function of concentration of synthetic myxopyronins A and B in anin vitro transcription assay using E. coli RNA polymerase.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a convergent synthetic route to preparemyxopyronin compounds, including myxopyronins A and B (FIG. 1). Theinvention provides two variant pathways based on alternative approachesto install the side chain at the pyrone 3-position.

Pathway A

Starting with 2-pentanone, a Wadsworth-Emmons reaction is performed withtriethyl phosphonoacetate in THF in the presence of NaH. Ester 16 (seeFIG. 3) is sequentially reduced with DIBAL and oxidized with DDQ toproduce aldehyde 14. A Wadsworth-Emmons reaction is used to condensealdehyde 14 and triethyl 2-phosphonopropionate in THF in the presence ofNaH. The resulting product is sequentially reduced with DIBAL andoxidized with DDQ to produce aldehyde 1. This constitutes the 3-positionside chain precursor.

The pyrone portion is constructed as follows. Ethyl propionylacetate ishydrolyzed with aqueous NaOH. Two equivalents of the resulting acid arecondensed with an equivalent of carbonyidiimidazole to produce pyrone 5(see FIG. 5). The 3-propionyl group is hydrolyzed using concentratedH₂SO₄ at elevated temperature to afford pyrone 6, which is thenalkylated with 3-bromopropionaldehyde dimethylacetal using n-BuLi andTHF/HMPT as a solvent to give 7. The 4-position hydroxyl is converted toSEM ether 8 using SEM-Cl and diisopropylamine in CH₂Cl₂. Thedimethylacetal is removed under acidic conditions and the resultingaldehyde is alkylated under Wadsworth-Emmons conditions withtriethylphosphonoacetate in THF in the presence of NaH to produce theunsaturated ester 9. The SEM ether is cleaved using TBAF and DMPU togive key intermediate 10.

Intermediate 10 can be synthesized by an alternate pathway. Pyrone 6 isdeprotonated with n-BuLi and then alkylated with allyl bromide inTHF/HMPT. The 4-position hydroxyl is converted to SEM ether 11 bytreatment with SEM-Cl and diisopropylamine in CH₂Cl₂. The allyl group ishydroborated using a borane reagent and the subsequent borate is removedoxidatively to give the alcohol. The resulting alcohol is oxidized understandard Swern conditions to give aldehyde 12. Aldehyde 12 was alkylatedwith triethylphosphonoacetate in THF in the presence of NaH to produceunsaturated ester 9. The SEM ether is removed under acidic conditions,e.g., using H₂SO₄ in THF/EtOH, to give the key intermediate 10. An aldolreaction between compounds 1 and 10 catalyzed by TFA in CH₂Cl₂ providesintermediate 13. The synthesis can be finished by oxidation to produceintermediate 17 followed by a Curtius sequence to produce myxopyronin A18.

Pathway B

The second route takes advantage of the convenient availability ofintermediates 10 and 14 by processes disclosed below. Pyrone 10 isacylated with propionyl chloride in TFA at elevated temperature toafford intermediate 15 (FIGS. 11a and 11 b). A base-catalyzed aldolbetween compounds 14 and 15 using LDA in THF forms an intermediate thatis sequentially treated with mesyl chloride and triethylamine in CH₂Cl₂followed by DBU to afford compound 17, which is also an intermediate inpathway A. Myxopyronin A results after a Curtius sequence.

The present invention provides a process of preparing a myxopyroninhaving the structure:

which comprises:

(a) condensing an aldehyde having the structure:

with a pyrone having the structure:

under suitable conditions to form an adduct having the structure:

(b) oxidizing the adduct formed in step (a) under suitable conditions toform a pyrone ketone having the structure:

and (c)(I) saponifying the pyrone ketone formed in step (b) undersuitable conditions to form a pyrone acid;

(ii) acylating the pyrone acid formed in step (c)(I) under suitableconditions to form a pyrone anhydride; and

(iii) treating the pyrone anhydride formed in step (c)(ii) with an azidesalt to form a pyrone acyl azide; and

(iv) heating the pyrone acyl azide formed in step (c)(iii) in methanolunder suitable conditions to form myxopyronin A.

As practiced in the present invention, the 1,2-addition step (a) recitedabove is performed using an acid catalyst, such as trifluoroacetic acid(TFA), hydrochloric acid, sulfuric acid, or p-toluenesulfonic acid,preferably in the presence of a dehydrating agent, such as molecularsieves, more preferably using 4Å molecular sieves, in an inert organicsolvent, such as dichloromethane or p-dioxane. Alcohol oxidation step(b) is carried out using various oxidants, such as2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), manganese dioxide orchromium chlorochromate. Saponifying step (c)(I) is carried out using ahydroxide base, such as LiOH, NaOH, KOH or CsOH, in a solvent mixturecomprising water and at least one organic co-solvent, such as methanol,ethanol, or tetrahydrofuran (THF). A preferred solvent composition ismethanol/THF/water in the proportion 2:2:1. In the Curtius sequence,step (c)(ii) is effected using an alkyl or aryl chloroformate,including, but not limited to, methyl chloroformate, ethylchloroformate, isopropyl and phenyl chloroformate, in the presence of abase, such as DIPEA, in an inert organic solvent, preferably misciblewith water, preferably, acetone. The resulting product may be purified,or used directly in the subsequent step. Step (c)(iii) is carried outusing an azide salt, such as lithium azide, sodium azide, ortetraalkylammonium azide, preferably in the presence of water.Rearrangement step (c)(iv) entails heating the acid azide formed in step(c)(iii) in a solvent mixture, containing an alcohol, such as ethanol,methanol, or phenol, preferably, methanol, and an inert solvent such asbenzene or toluene, at elevated temperatures, preferably, at the refluxtemperature of the solvent mixture.

The present invention also provides a process of preparing a myxopyroninhaving the structure:

which comprises:

(a) treating a pyrone having the structure:

with propionyl chloride under suitable conditions to form a ketoneadduct having the structure:

(b)(I) condensing the ketone adduct formed in step (a) under suitableconditions with an aldehyde having the structure:

to form a pyrone aldol; (ii) mesylating the pyrone aldol formed in step(b)(I) under suitable conditions to form a pyrone aldol mesylate; and(iii) reacting the pyrone aldol mesylate under suitable basic conditionsto form a pyrone ketone having the structure:

and (c)(I) saponifying the pyrone ketone formed in step (b)(iii) undersuitable conditions to form a pyrone acid;

(ii) acylating the pyrone acid formed in step (c)(I) under suitableconditions to form a pyrone anhydride; and

(iii) treating the pyrone anhydride formed in step (c)(ii) with an azidesalt to form a pyrone acyl azide, and (iv) heating the pyrone acyl azideformed in step (c)(iii) under suitable conditions to form themyxopyronin.

Acylation step (a) may be effected using an acid catalyst, such as TFA,hydrochloric acid, or p-toluenesulfonic acid, preferably in the presenceof a dehydrating agent, such as molecular sieves, preferably using 4 Åmolecular sieves, in an inert organic solvent, such as dichloromethane.Condensation step (b)(i) is performed using a strong, non-nucleophilicbase, such as LDA, potassium t-butoxide, or sodium hydride, preferably,LDA, in an inert organic solvent, such as THF. Mesylation step (b)(ii)is carried out using an acylating agent, such as mesyl chloride, p-tosylchloride, acetic anhydride, preferably, mesyl chloride, in the presenceof a non-nucleophilic organic base, such as DIPEA or triethylamine.Elimination step (b)(iii) may be performed using a variety of reagentseffective to cause elimination, preferably, a strong non-nucleophilicbase such as DBU. The Curtius rearrangement is effected as describedabove.

The present invention also provides a process of preparing anunsaturated aldehyde having the structure:

which comprises:

(a) condensing triethyl phosphonoacetate with 2-pentanone under suitableconditions to form an unsaturated ester having the structure:

(b)(i) reducing the unsaturated ester formed in step (a) under suitableconditions to form an unsaturated alcohol; and (ii) oxidizing theunsaturated alcohol under conditions suitable to form a monounsaturatedaldehyde having the structure:

and (c)(i) condensing triethyl phosphonopropionate with themonounsaturated aldehyde formed in step (b)(ii) under suitableconditions to form a diene ester;

(ii) reducing the diene ester formed in step (c)(i) under suitableconditions to form a diene alcohol; and

(iii) oxidizing the unsaturated alcohol under suitable to form theunsaturated aldehyde.

As implemented in the present invention, the condensation step,preferably of the Wadsworth-Emmons type, is favorably performed in thepresence of a non-nucleophilic base, such as LDA or sodium hydride, inan inert polar, aprotic organic solvent, such as THF. The esterreduction step (b)(i) may be carried out using any of a variety ofreducing agents, such as diisobutylaluminum hydride (DIBAL) in an inertaprotic organic solvent, such as THF. Oxidation step (b)(ii) may beeffected using any of a range of mild oxidants, preferably, DDQ, in aninert organic solvent, such as dichloromethane. The subsequentcondensation step (c)(i) may be performed in the presence of anon-nucleophilic base, such as sodium hydride, in an inert organicsolvent, preferably, THF. Reduction step (c)(ii) may be carried outusing various reducing agents, preferably, diisobutylaluminum hydride(DIBAL) in an aprotic organic solvent, such as THF. Finally, oxidationstep (c)(iii) may be effected using a variety of oxidants, such as DDQ,in dichloromethane.

In one embodiment, the present invention provides a process of preparingthe unsaturated ester having the structure:

which comprises (a) condensing ethyl butyryl acetate with diethylphosphorochloridate under suitable conditions to form an enolphosphonate; and (b) alkylating the enol phosphonate formed in step (a)with an organometallic reagent under suitable conditions to form theunsaturated ester.

As practiced in the invention, condensing step (a) is performed using anon-nucleophilic base, including, but not limited to, potassiumt-butoxide, sodium hydride, LDA, and lithium diethylamide, preferably,sodium hydride, in an inert aprotic organic solvent, such as THF ordiethyl ether. Alkylation step (b) is effected using an organometallicreagent, such as lithium dimethyl cuprate, methyl lithium, methylmagnesium bromide or chloride, preferably, lithium dimethyl cuprate, inan inert aprotic solvent, preferably, diethyl ether, at a temperatureranging from about —100° C. to about 0° C., more preferably, from about−90° C. to about −50° C., most preferably, at −78° C.

In addition, the present invention provides a process of preparing apyrone ester having the structure:

which comprises:

(a) treating a dicarbonyl compound having the structure:

acidifying and dimerizing under suitable conditions to form an acylpyrone having the structure:

(b) hydrolyzing the acyl pyrone formed in step (a) under suitableconditions to form a pyrone having the structure:

(c) alkylating the pyrone formed in step (b) under suitable conditionsto form a pyrone acetal having the structure:

(d) etherifying the pyrone acetal formed in step (c) under suitableconditions to form an ether pyrone acetal having the structure:

(e) (i) acidolytically cleaving the pyrone acetal under suitableconditions; and (ii) reacting with triethyl phosphonoacetate undersuitable conditions to form a protected pyrone ester having thestructure:

and (f) deprotecting the protected pyrone ester under suitableconditions to form the pyrone ester.

Hydrolysis in step (a) recited above is carried out using a hydroxidebase, such as LiOH, NaOH, or KOH, preferably NaOH, followed byacidification with a variety of acids, including, but not limited to,hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, and TFA,preferably, hydrochloric acid. Dimerization in step (a) is effectedusing a condensing agent, such as carbonyl diimidazole, or an equivalentreagent known in the art. Hydrolysis step (b) is performed using astrong acid such as hydrochloric acid, hydrobromic, trifluoroacetic orsulfuric acid, preferably 90% sulfuric acid, at elevated temperature, inthe range from about 65° C. to about 160° C., preferably between about100° C. and about 140° C., more preferably, at 130° C. Alkylation step(c) with fluor-, Clair- or bromopropionaldehyde dimethyl acetal iscarried out using a strong base such as n-butyl lithium, t-butyllithium, sec-butyl lithium or phenyl lithium, in an inert polar aproticsolvent, such as THF in the presence or absence of a co-solvent, such asHMPT. Etherification step (d) is effected using a strongnon-nucleophilic base such DIPEA or triethylamine in an inert organicsolvent, such as dichloromethane. Acetal cleavage step (e)(i) is carriedout using a strong acid, such as sulfuric acid or p-tonic acid, in asolvent mixture preferably comprising THF and water in a ratio of 10:1.Condensation step (e)(ii) employs a strong non-nucleophilic base, suchas sodium hydride, in a solvent mixture favorably comprising toluene andDMF. Deprotection step (f) utilizes an organic fluoride salt, preferablytetrabutylammonium fluoride.

The present invention further provides a process of preparing a pyroneester having the structure:

which comprises:

(a) (i) alkylating a pyrone having the structure:

(ii) etherifying the pyrone formed in step (i) under suitable conditionsto form protected pyrone having the structure:

(b)(i) hydroborating the protected pyrone formed in step (a)(ii) undersuitable basic conditions; and (ii) oxidizing under suitable conditionsto form a pyrone aldehyde having the structure:

(c) condensing the pyrone aldehyde formed in step (b)(ii) with triethylphosphonoacetate under suitable conditions to form a protected pyroneester having the structure:

and (d) cleaving the protected pyrone ester under suitable conditions toform the pyrone ester.

As practiced in the invention, allylation step (a)(i) is effected usingallyl chloride, bromide or fluoride as the halide and a strong base,such as sodium hydride or n-butyl lithium, in a polar aprotic solventmixture favorably made up of THF and HMPT. Protection step (a)(ii) iscarried out using a strong non-nucleophilic base, such as DIPEA ortriethylamine, in an inert organic solvent, such as dichloromethane.Hydroboration (b)(i) is performed with a borane complex, such asBH₃—SMe₂, followed by treatment with hydrogen peroxide and inorganicbase, such as NaOH. Oxidation (b)(ii) preferably utilizes standard Swernconditions. Condensation step (c) is performed using a strongnon-nucleophilic base such as sodium hydride. Deprotection step (d) iseffected using a strong acid such as sulfuric acid in the presence of aprotic solvent, such as ethanol, and a miscible aprotic organic solvent,such as THF.

Generally, the Wadsworth-Emmons reaction is applied in the pathwayleading to both 1 and 9, and tolerates the use of aprotic, anhydroussolvents. While both toluene and THF have been used effectively, higheryields are obtained with THF. The preferred base is NaH although otheranhydrous bases are also effective. Other reactions generatingcarbon-carbon double bonds could be equivalently used. Intermediateester 16 may alternatively be prepared by an addition reaction involvinglithium dimethyl cuprate, or an equivalent organometallic reagent. Thereductions of the two esters on the pathway to 1 are both performed withDIBAL, although a number of hydride reagents could have been used. Anyaprotic, anhydrous solvent would be permissible as long as its meltingpoint is below −78° C. The reaction proceeds rapidly at −78° C., but mayoptionally be carried out at temperatures up to −40° C. without untowardconsequences. The oxidations on the pathway to 1 have been efficientlyperformed both under Swern conditions and with DDQ, or any reagentproficient in the oxidation of allylic alcohols or primary alcohols,including, but not limited to, manganese oxide, pyridiniumchlorochromate, and pyridinium dichromate. The pathway using DDQ is moreeffective with unsaturated alcohols. The oxidation reaction ispreferably performed using a variety of aprotic, anhydrous solvents.

With respect to the production of the pyrone, ethyl propionylacetate ishydroyzed with aqueous NaOH. The reaction may be carried out using anyhydroxylic base. The dimerization of the acid to produce pyrone 5 ispreferably effected in aprotic, anhydrous solvents. Othercarbonyldiimidazole equivalents (e.g., phosphene) can be similarly used.The hydroysis of the propionyl groups to yield 6 occurs in strong acids,including, but not limited to, hydrochloric acid, phosphoric acid,nitric acid. The alkylation of 6 with either 3-bromopropionaldehydedimethyl acetal or allyl bromide is preferably performed in the presenceof hexamethylphosphoric triamide (HMPT) to help solubilize the anion.

The SEM ether is a preferred protecting group. For the installationstep, various amine bases may be used, including, but not limited to,diisopropylethylamine (DIPEA), triethylamine, DBU(1,8-diazbicyclo[5.4.0]undec-7-ene), and pyridine. The reaction may beperformed in various polar aprotic solvents, including, but not limitedto, chloroform, carbon tetrachloride and dimethyl formamide (DMF). Theremoval of the dimethyl acetal using dilute H₂SO₄ in THF/H₂O may becarried out with mild acids which do not cleave the SEM group. Ingeneral, if H₂O is used as a co-solvent rather than an alcohol, the SEMgroup is not affected. The removal of the SEM group in intermediate 9 toproduce compound 10 is effected using either TBAF/DMPU or dilute H₂SO₄in THF/EtOH, acidic conditions being the more effective.

The hydroboration of intermediate 11 is carried out with a number ofborane reagents, e.g., BH₃.THF or borane-dimethyl sulfide complex (FIG.7). Yields are limited by the reactivity of the pyrone under theseconditions. The aldol reaction between compounds 1 and 10 is bestperformed using TFA in CH₂Cl₂. In the alternate route used to attach the3-position side chain, the acylation of compound 10 with propionylchloride to produce intermediate 15 proceeds efficiently in the presenceof TFA at elevated temperature. Lithium diisopropylamide (LDA) is anefficient base for the aldol reaction of between compounds 14 and 15yielding intermediate 17. A variety of other bases are also useful forthe purpose, including, but not limited to, hydroxide bases,lithium-containing bases and alkoxides.

The present invention provides a process of preparing a myxopyroninhaving the structure:

which comprises:

(a) treating an acyl pyrone having the structure:

with an unsaturated aldehyde having the structure:

under suitable conditions to form a pyrone aldol having the structure:

(b)(i) mesylating the pyrone aldol formed in step (a) under suitableconditions to form a pyrone aldol mesylate; and (ii) reacting the pyronealdol mesylate formed in step (b)(i) under suitable basic conditions toform a pyrone diene having the structure:

and (c) saponifying the pyrone diene formed in step (b)(ii) undersuitable conditions to form a pyrone acid; (d) converting the pyroneacid formed in step (c) under suitable conditions to a pyrone acylazide; and (e) solvolyzing the pyrone acyl azide formed in step (d) withmethanol under suitable conditions to form the myxopyronin.

In one embodiment, the present invention provides a process as disclosedabove wherein the acyl pyrone in step (a) is treated with theunsaturated aldehyde in the presence of a Lewis acid catalyst. In acertain embodiment, the present invention provides a process wherein theacid catalyst is titanium tetrachloride/triethylamine combination. Inanother embodiment, the present invention provides a process asdisclosed above wherein the pyrone aldol in step (b)(i) is mesylatedwith methanesulfonyl (mesyl) chloride in the presence of triethylamine.In another embodiment, the present invention provides a process asdisclosed above wherein the pyrone aldol mesylate in step (b)(ii) isreacted with DBU. In another embodiment, the present invention providesa process as above wherein the pyrone diene in step (c) is saponifiedwith lithium hydroxide. In yet another embodiment, the present inventionprovides a process as above wherein the pyrone acid in step (d) isconverted using diphenylphosphoryl azide in the presence oftriethylamine.

The aldol condensation of step (a) may be effected at subambienttemperatures, preferably at −78° C. Mesylation step (b)(i) is carriedout preferably using mesyl chloride but an equivalent reaction sequencewould result using another acylating agent, such as p-tosyl chloride,acetic anhydride. Step (b)(i) occurs efficiently in the presence of anon-nucleophilic organic base, such as DIPEA or triethylamine.Elimination step (b)(ii) may be performed using a reagent effective tocause elimination, preferably, a strong non-nucleophilic base such asDBU. Saponifying step (c) is effected using a base such as lithiumhydroxide, sodium hydroxide or potassium hydroxide under aqueous ormixed aqueous/dipolar solvent conditions. Converting step (d) is carriedout by heating the pyrone acid with diphenylphosphoryl azide, or anequivalent reagent, in the present of a strong non-nucleophilic base,such as triethylamine or diethylisopropylamine, in a noninteractingorganic solvent, such as benzene, toluene or xylene, at a temperatureabove room temperature, preferably at the reflux temperature of thesolvent. Solvolyzing step (e) is effected by heating the pyrone acylazide in methanol at a temperature above room temperature, preferably atthe reflux temperature of methanol.

The present invention provides a process of preparing an acyl pyronehaving the structure:

which comprises:

(a) oxidizing a pyrone diol having the structure:

under suitable oxidizing conditions to form a pyrone aldehyde having thestructure:

and (b) condensing the pyrone aldehyde formed in step (a) withtriethylphosphonoacetate under suitable basic conditions to form thepyrone ester. In one embodiment, the present invention provides aprocess as shown above wherein the pyrone diol in step (a) is oxidizedwith a Dess-Martin periodinate. In another embodiment, the presentinvention provides a process as above wherein the condensing step iseffected with sodium hydride.

Oxidizing step (a) is carried out using any oxidant known in the artsuited to the purpose, including chromium oxide, pyridiniumchlorochromate, dicyclo-hexylcarbodiimide/dimethylsulfoxide, aluminumoxide/acetone, lead tetra acetate, etc. A preferred oxidant is theDess-Martin periodinate. Condensing step (b) is effected in the presenceof a strong non-nucleophilic base, such as sodium hydride or potassiumt-butoxide, in an inert organic solvent such as benzene, toluene, orxylene, typically at ambient temperatures.

The present invention provides a process of preparing a pyrone diolhaving the structure:

which comprises:

(a)(i) deprotonating an acyl pyrone having the structure:

under suitable basic conditions to form an anion; and

(ii) alkylating the anion formed in step (a)(i) with a siloxyalkylhalide having the structure:

under suitable conditions to form an alkylated pyrone having thestructure:

and (b) deprotecting the alkylated pyrone formed in step (a)(ii) undersuitable conditions to form the pyrone diol. In one embodiment, thepresent invention provides a process as disclosed wherein the acylpyrone in step (a)(i) is deprotonated using lithium diisopropylamide orlithium diethylamide. In another embodiment, the present inventionprovides a process as above wherein the alkylated pyrone in step (b) isdeprotected with a weak acid. In a certain embodiment, the inventionprovides a process wherein the weak acid is acetic acid.

Deprotonating step (a)(i) is carried out using a strong non-nucleophilicbase, such as LDA, lithium diethylamide, potassium t-butoxide, sodiumamide, sodium hydride, etc., in a non-interacting organic dipolarsolvent or solvent mixture, such as tetrahydrofuran (THF) and/orhexamethylphosphoramide (HMPA), at subambient temperatures, preferablyat −78° C. The deprotonated dianion is used directly in the alkylationstep (a)(ii) is performed at subambient temperatures, preferably −78° C.

The present invention provides a process of preparing a myxopyroninhaving the structure:

wherein R is C₁₋₉alkyl, and wherein R₁ is C₁₋₉alkoxy; which comprises:

(a) condensing an aldehyde having the structure:

with a pyrone having the structure:

wherein R₀ is C₁₋₉alkyl, under suitable conditions to form a pyroneketone having the structure:

and (b)(i) saponifying the pyrone ketone formed in step (a) undersuitable conditions to form a pyrone acid; and (ii) treating the pyroneacid under suitable Curtius conditions to form the myxopyronin.

In one embodiment, the subject invention provides a process as abovewherein the pyrone is condensed with the aldehyde in the presence of atitanium(IV) reagent. In another embodiment, the pyrone ketone issaponified in the presence of a hydroxide salt. For example, thehydroxide salt is LiOH, NaOH, KOH, ammonium hydroxide,tetramethylammonium hydroxide, tetraethyl-ammonium hydroxide,tetra-n-propylammonium hydroxide or tetra-n-butylammonium hydroxide. Ina certain embodiment, the present invention provides a process as abovewherein the Curtius conditions comprise:

(a) acylating the pyrone acid to form a pyrone anhydride;

(b) treating the pyrone anhydride formed in step (a) with an azide saltto form a pyrone acyl azide; and

(c) heating the pyrone acyl azide formed in step (b) with an alcoholR₁OH under conditions suitable to form the myxopyronin. In oneembodiment, the process is carried out using methanol as the alcoholR₁OH. In another embodiment, the pyrone is treated with alkylhaloformate, and subsequently with an azide salt. The alkylhaloformatemay favorably be methyl or ethyl chloroformate, and the azide salt maybe LiN₃ or NaN₃. In addition, R may be methyl or ethyl. The presentinvention also provides a process of preparing an unsaturated aldehydehaving the structure:

wherein R is C₁₋₉alkyl; which comprises:

(a) treating an acetylene having the structure:

R—≡

with a first organometallic reagent to form a first intermediate;

(b) reacting the first intermediate with a second organometallic reagentso as to form a second intermediate comprising a reactive(E)-trisubstituted vinylaluminate;

(c) condensing the second intermediate with paraformaldehyde undersuitable conditions to form an allylic alcohol having the structure:

and (d) oxidizing the allylic alcohol formed is step (c) under suitableconditions to form the unsaturated aldehyde.

In one embodiment, the process may be effected wherein the firstorganometallic reagent comprises a zirconocene dihalide in the presenceof a trialkylalane. In particular, the zirconocene dihalide iszirconocene dichloride and the trialane is trimethylaluminum. In anotherembodiment, the process may be effected wherein the secondorganometallic reagent is an alkyllithium reagent. In this process, theallylic alcohol is oxidized with any of a variety of oxidants suited forthe purpose, for example, pyridinium chlorochromate, pyridinedichloride, manganese dioxide, a Swern reagent or tetrapropylammoniumperruthenate in the presence of N-methylmorpholine N-oxide.

In treating step (a) disclosed above, the first organometallic reagentis typically prepared from a metallocene dihalide and a trialkylaluminumreagent, using an organic-miscible solvent such as dichloromethane ordichloroethane, at subambient temperatures, preferably at −5° C. to 5°C., more preferably at 0° C. The addition of the acetylenic reagent maybe effected at a temperature between −20° C. and 30° C., preferably at15° C. to 25° C., more preferably at room temperature. Prior to step(b), solvents are evaporated and the residue is extracted with ahydrocarbon solvent, such as n-pentane, n-hexanes or heptane. Thisextract is then treated with the second organometallic reagent, forexample, an alkyl lithium such as n-butyl lithium, typically atsubambient temperatures, preferably at 0° C. Condensing step (c) may becarried out by transferring the solution of the second intermediate to asuspension of paraformaldehyde in a non-aqueous dipolar solvent such asTHF, at a temperature ranging from 0° C. to 35° C., but preferably atroom temperature.

The present invention further provides a process of preparing amyxopyronin having the structure:

wherein R is C₁₋₉alkyl, and wherein R₁ is NH₂, alkylamine, dialkylamine,or optionally substituted phenylamine; which comprises:

(a) saponifying a pyrone ketone having the structure:

wherein R₀ is C₁₋₉alkyl, under suitable conditions to form a pyroneacid; and (ii) treating the pyrone acid under suitable Curtiusrearrangement conditions to form the myxopyronin. In one embodiment, thepyrone ketone is saponified in the presence of a hydroxide salt. Thehydroxide salt may be LiOH, NaOH, KOH, ammonium hydroxide,tetramethylammonium hydroxide, tetraethylammonium hydrox-ide,tetra-n-propylammonium hydroxide or tetra-n-butyl-ammonium hydroxide.The process is favorably carried out wherein the Curtius conditionscomprise:

(a) acylating the pyrone acid to form a pyrone anhydride;

(b) treating the pyrone anhydride formed in step (a) with an azide saltto form a pyrone acyl azide; and

(c) treating the pyrone acyl azide formed in step (b) with an ammonia,alkylamine, dialkylamine or optionally substituted phenylamine underconditions suitable to form the myxopyronin. In one embodiment, thealkylamine is methylamine. In another embodiment, the pyrone is treatedwith an alkyl haloformate, and subsequently with an azide salt. Inparticular, the alkylhaloformate is favorably methyl or ethylchloroformate, and the azide salt is LiN₃ or NaN₃. The process may becarried out wherein R is methyl or ethyl.

In saponifying step (a) the pyrone ketone is dissolved in a nonaqueousdipolar solvent such as THF and treated with a hydroxide salt such aslithium hydroxide, sodium hydroxide, or potassium hydroxide, preferablylithium hydroxide, at a temperature ranging from 0° C. to 45° C., andpreferably at room temperature. In treating step (b) the azide salt maybe any suitable azide salt, such as trimethyammonium azide, lithiumazide, sodium azide or potassium azide, which is dissolved water with orwithout a miscible non-reactive cosolvent prior to addition to theproduct of step (a), favorably at subambient temperatures, preferably at0° C. Treating step (c) is carried out in a non-reactive dipolar solventsuch as THF at a temperature determined by the ammonia or aminecomponent reacted, and may range from −78° C. to 102° C.

A composition of matter having the structure:

wherein R is C₁₋₉alkyl, and wherein R₁ is H, C₁₋₉alkyl, benzyl,optionally substituted phenyl, OH, C₁₋₉alkoxy, NH₂, alkylamine,dialkylamine, or optionally substituted phenylamine.

The present also provides a composition of matter having the structure:

wherein R is C₁₋₉alkyl, and wherein R₁ is H, C₁₋₉alkyl, benzyl,optionally substituted phenyl, OH, C₁₋₉alkoxy, NH₂, alkylamine,dialkylamine, or optionally substituted phenylamine; and wherein when Ris methyl or ethyl, R₁ is not methoxy.

The present invention further provides the following compositions ofmatter, having the structures set forth below. These compounds areuseful as intermediates in the synthesis of myxopyronins andcorallopyronins according to the present invention:

The scope of the present invention includes compositions of matterwherein the C_(α) carbon at the 6-position of the pyrone ring thereinpossesses either an R or S absolute configuration, as well as mixturesthereof. The processes of the present invention encompass the use ofvarious alternate protecting groups known in the art. For example, inthe preparation of unsaturated aldehyde 1, ethyl ester 16 may beequivalently replaced with a methyl, propyl, isobutyl, phenyl or benzylester, wherein the triethylphosphonoacetate ortriethylphosphonopropionate may be replaced with the correspondingalternate ester. Similarly, in the preparation of intermediate pyroneester 10 and of myxopyronin 18, the ethyl ester may be equivalentlyreplaced with, for example, a methyl, propyl, isobutyl, phenyl, orbenzyl ester, wherein the triethylphos-phonoacetate used in theconversion from 8 to 9 may be replaced with the corresponding alternateester. Furthermore, in the conversion from compound 6 to 7, thebromopropionaldehyde dimethyl acetal may be equivalently replaced with,for example, an ethyl, propyl, butyl, ethylene, or propylene acetal, andin the conversion from 7 to 8, SEM-Cl may be equivalently replaced withanother protecting group, for example, methoxymethyl, methyl-thiomethyl,trimethylsilyl, t-butyidimethylsilyl, or tetrahydropyranyl.

The present invention will be better understood from the ExperimentalDetails which follow. However, one skilled in the art will readilyappreciate that the specific methods and results discussed are merelyillustrative of the invention as described in the claims which followthereafter.

General

¹H and ¹³C NMR spectra were taken in CDCl₃ at 400 MHz and 75 MHzrespectively unless specified otherwise. Chemical shifts are reported inparts per million using the solvent resonance internal standard(chloroform, 7.24 and 77.0 ppm respectively, unless specifiedotherwise). NMR data are reported as follows: chemical shift,multiplicity (app=apparent, par obsc=partially obscured,ovrlp=overlapping, s=singlet, d-doublet, t=triplet, q=quartet,m=multiplet, br=broad, abq=ab quartet), coupling constant, andintegration. Infrared Resonance (IR) spectra were recorded on aPerkin-Elmer 1800 series FTIR spectrophotometer. High resolution massspectra were obtained on a Finnegan MAT-90 spectrometer in the BostonUniversity Mass Spectrometry Laboratory. Reversed phase preparative HPLCwas conducted on a Varian/Rainin SD-200 equipped with Dynamax PDA-2Diode Array detector, using 22×250 mm Vydac C18 column (218TP1022).Methylene chloride (CH₂Cl₂), methanol (MeOH), benzene (C₆H₆), toluene,and hexane were distilled from calcium hydride, and tetrahydrofuran(THF) and hexamethyl phosphoramide (HMPA) were distilled from sodium andbenzophenone prior to use. Titanium tetrachloride (TiCl₄) was freshlydistilled from copper powder under reduced pressure before each use.Anhydrous 1,2-dichloro ethane (ClCH₂CH₂Cl), trimethyl aluminum (AlMe₃,2.0 M solution in hexanes) and zirconocene dichloride (Cp₂ZrCl₂) waspurchased from Aldrich Chemical Company Inc. and used without furtherpurification. All other reagents were used as supplied. All reactionswere carried out in oven-dried glassware under argon atmosphere unlessotherwise noted. Analytical thin layer chromatography was performed onWhatman Reagent 0.25 mm silca gel 60-A plates. Flash chromatography wasperformed on E. Merck silica gel 230-400 mesh.

EXAMPLE 1 Synthesis of 6-Ethyl Pyrone Hydrolysis of the EthylPropionylacetate 4

To a flame-dried flask under flushing argon containing 25 g (175 mmol)of ethyl propionylacetate 4 was added 300 mL of 1.5M NaOH. The solutionwas stirred at room temperature for 18 hours. TLC indicated that thestarting material was gone. The solution was placed in an ice/H₂O bathand concentrated HCl was added until the pH of the solution was 1. Thereaction was allowed to warm to room temperature. The solution wassaturated with KCl and extracted with EtOAc (3×100 mL) and CHCl₃ (3×100mL). The combined organic layers were dried with Na₂SO₄, filtered andevaporated on a rotary evaporator. This yielded 15.5 g (76%) of a whitesolid. The product needed no further purification and was used directlyi n the next step.

¹H NMR (300 MHz, CDCl₃): δ1.1 (3H, t); 2.6 (2H, q); 3.5 (2H, s).

Dimerization of the acid to produce the pyrone 5

To a flame-dried flask under flushing argon containing 10.55 g (91 mmol,1 eq) of the acid dissolved in 250 mL of freshly distilled THF was added16.22 g (100 mmol, 1.1 eq) of carbonyldiimidazole. The reaction was leftto stir for 12 hours. The reaction was concentrated using a rotaryevaporator. The residue was partitioned between 100 mL of CHCl₃ and 100mL of 10% HCl. The aqueous layer was extracted with CHCl₃ (2×50 mL). Thecombined organic layers were dried with Na₂SO₄, filtered and evaporatedon a rotary evaporator. The product needed no further purification aslong as it was left on a vacuum pump long enough to remove any remainingstarting acid. This yielded 8.8 g (98%) of 5 a tan solid.

¹H NMR (300 MHz, CDCl₃): δ1.15 (3H, t); 1.25 (3H, t); 2.55 (2H, q); 3.1(2H, q); 5.95 (1 H, s).

Removal of the propionoyl group to produce 6

In a round-bottomed flask was placed 11.4 g (58.2 mmol, 1 eq) of thepyrone 5 and it was dissolved in 50 mL of concentrated H₂SO₄. Thesolution was heated to 130° C. in an oil bath for 15 minutes. Thereaction was allowed to cool to room temperature. Approximately 50 g ofice was added with stirring. The solution was extracted with Et₂O (3×50mL). The combined organic layers were dried with Na₂SO₄, filtered andevaporated on a rotary evaporator. This yielded 7.7 g (94%) of 6 a tansolid. The product needed no further purification.

¹H NMR (300 MHz, CDCl₃): δ1.2 (3H, t); 2.5 (2H, q); 5.65 (1H, d); 6.0(1H, d).

EXAMPLE 2 Construction of 6-position side chain 3-Bromopropionaldehydedimethyl acetal approach Alkylation of pyrone with3-bromopropionaldehyde dimethyl acetal to produce 7

To a flame-dried flask under flushing argon containing 2.5 g (18 mmol, 1eq) of the ethyl pyrone 6 dissolved in 40 mL of freshly distilled THFwas added 8 mL of HMPT. The solution was slowly cooled to −78° C. in adry ice/acetone bath, making sure the ethyl pyrone stayed in solution.Once the temperature had equilibrated, 24.6 mL (39 mmol, 2.2 eq) of 1.6M n-BuLi was added by syringe. The solution quickly became a marooncolor. The dry ice/acetone bath was replaced with an ice/H₂O bath andthe solution allowed to stir for 30 minutes. At this point, the3-bromopropionaldehyde dimethyl acetal was added by syringe. Thesolution was left to stir and warm to room temperature overnight. Thereaction was quenched by the addition of 25 mL of H₂O. Addition of 1%HCl occurred until the solution was acidic. The solution was extractedwith Et₂O (3×50 mL). The combined organic layers were extracted withsaturated brine solution, dried with Na₂SO₄, filtered and evaporated ona rotary evaporator. The product was 7 a brown oil (1.75 g) and was notpurified at this time, and was used directly in the production of theSEM ether.

Conversion of the 4-hydroxyl group of 7 into the SEM ether 8

To a flame-dried flask under flushing argon containing 1.75 g (12 mmol,1 eq) of the crude alkylation product 7 dissolved in 40 mL of anhydrousCH₂Cl₂ was added 2.42 mL (12 mmol, 1 eq) of N,N-diisopropylethylamine(DIPEA) by syringe. The solution immediately became orange. The solutionwas cooled with a ice/H₂O bath and 2.46 mL (12 mmol, 1 eq) of2-(trimethylsilyl)ethoxymethyl chloride (SEM-Cl) was added by syringe. Awhite vapor formed. The reaction was allowed to stir for 2 hours atwhich time TLC showed that no starting material remained (5:2 benzene/ethyl acetate). The reaction was diluted with 150 mL of Et₂O and theresulting solution extracted with saturated NaHCO₃ (2×50 mL). Theorganic layer was extracted with brine solution (1×50 mL). The organiclayer was dried with Na₂SO₄, filtered and evaporated on a rotaryevaporator. The residue was placed on a SiO₂ column and eluted with 5:2benzene/ethyl acetate. This yielded 0.46 g (7% for the two stepscombined) of a light yellow oil 8.

¹H NMR (300 MHz, CDCl₃): δ0.0 (9H, s); 1.0 (2H, t); 1.2 (3H, d); 1.5-1.8(4H , m); 2.5 (1H, m); 3.3 (6H, 2s); 3.7 (2H, t); 4.3 (1H, t); 5.2 (2H,s); 5.6 (1H, d)

Deprotection of the aldehyde

In a round-bottomed flask was placed 0.45 g (1.2 mmol, 1 eq) of thepyrone 8 and it was dissolved in 22 mL of a 10:1 mixture of THF/H₂O. Tothis solution was added 10 drops of concentrated H₂SO₄ and the reactionleft to stir. After 3 hours, 3 more drops of H₂SO₄ were added. After 3additional hours, TLC showed no more starting material (5:2benzene/ethyl acetate). The reaction was diluted with 20 mL of Et₂O andthis solution extracted with saturated NaHCO₃ (1×10 mL) and brine (1×10mL). The organic layer was dried with Na₂SO₄, filtered and evaporated ona rotary evaporator. This yielded 0.39 g (>98%) of a light yellow oil12. The product needed no further purification.

¹H NMR (300 MHz, CDCl₃): δ0.0 (9H, s); 1.0 (2H, t); 1.2 (3H, d); 1.8-2.0(4H, m); 2.6 (1H, m); 3.7 (2H, t); 5.2 (2H, s); 5.6 (1H, d); 5.8 (1H, d)9.75 (1H, t).

Horner-Emmons-Wadsworth reaction of the deprotected aldehyde to produce9

To a flame-dried flask under flushing argon containing 0.236 mL (1.2mmol, 1 eq) of the triethyl phosphonoacetate dissolved in 15 mL offreshly distilled toluene was added 50 mg (1.24 mmol, 1.05 eq) of NaH asa 60% mineral oil dispersion. Hydrogen evolution was witnessed. Oncethis subsided, 0.40 g (1.2 mmol, 1 eq) of the aldehyde dissolved intoluene (3 mL) was added by syringe. The syringe was washed with anadditional 2 mL of toluene and the solution added to the reaction.Almost immediately, an orange oil came out of solution. The reaction wasleft to stir overnight. The reaction was stopped by the addition of 20mL of H₂O and the solution made acidic with 1% HCl. The solution wasextracted with Et₂O (3×10 mL). The combined organics were extracted withsaturated NaHCO₃ (1×10 mL) and brine (1×10 mL). The organic layer wasdried with Na₂SO₄, filtered and evaporated on a rotary evaporator. Theresidue was placed on a SiO₂ column and eluted with 2:1 hexane/ ethylacetate. This yielded 0.347 g (74%) of a clear oil 9.

¹H NMR (300 MHz, CDCl₃): δ0.0 (9H, s); 0.9 (2H, t); 1.25 (3H, d); 1.25(3H, t); 1.6 (2H, m); 1.9 (1H, m); 2.2 (2H, m); 2.6 (1H, m); 3.7 (2H,t); 4.2 (2H, q); 5.2 (2H, s); 5.6 (1H, d); 5.8 (1H, d); 5.85 (1H, d);6.9 (1H, dt).

EXAMPLE 3 Allyl bromide approach Allylation of ethyl pyrone 6

To a flame-dried flask under flushing argon containing 0.108 g (0.77mmol, 1 eq) of the ethyl pyrone 6 dissolved in 20 mL of freshlydistilled THF was added 2 mL of HMPT. The solution was slowly cooled to−78° C. in a dry ice/acetone bath, making sure the ethyl pyrone stayedin solution. Once the temperature had equilibrated, 1.06 mL (1.7 mmol,2.2 eq) of 1.6 M n-BuLi was added by syringe. The solution quicklybecame a maroon color. The dry ice/acetone bath was replaced with anice/H₂O bath and the solution allowed to stir for 2 hours. At thispoint, the allyl bromide was added by syringe. As the end of theaddition was reached, the color of the solution became almost yellow.The reaction was allowed to stir for 1 hour. The contents of thereaction were partitioned between Et₂O and 1N HCl (25 mL of each). Thelayers were separated and the aqueous was extracted with Et₂O (2×25 mL).The organics were combined and extracted with 25 mL of saturated KCl.The organic layer was dried with Na₂SO₄, filtered and evaporated on arotary evaporator. This yielded 0.110 g (76%) of a light yellow oil,which needed no further purification.

¹H NMR (300 MHz, CDCl₃): δ1.2 (3H, d); 2.25 (1H, m); 2.45 (1H, m); 2.6(1H, m); 5.0 (1H, s); 5.05 (1H, d); 5.6 (1H, d); 5.7 (1H, m); 5.9 (1H,d).

Conversion of the 4-hydroxyl group into the SEM ether 11

To a flame-dried flask under flushing argon containing 0.110 g (0.61mmol, 1 eq) of the crude alkylation product dissolved in 3 mL ofanhydrous CH₂Cl₂ was added 0.106 mL (0.61 mmol, 1 eq) ofN,N-diisopropylethylamine (DIPEA) by syringe. The solution, whichimmediately became orange, was cooled with a ice/H₂O bath and 0.108 mL(0.61 mmol, 1 eq) of 2-(trimethylsilyl)ethoxymethyl chloride (SEM-Cl)was added by syringe whereupon a white vapor formed. The reaction wasallowed to stir for 2 hours at which time TLC showed that no startingmaterial remained (3:1 hexane/ethyl acetate). The reaction was dilutedwith 15 mL of Et₂O and the resulting solution extracted with saturatedNaHCO₃ (2×5 mL). The organic layer was extracted with brine solution (×5mL). The organic layer was dried with Na₂SO₄, filtered and evaporated ona rotary evaporator. The residue was placed on a SiO₂ column and elutedwith 3:1 hexane/ethyl acetate. This yielded 0.83 g (45%) of 11 as aclear oil.

¹H NMR (300 MHz, CDCl₃): δ0.0 (9H, s); 0.9 (2H, t); 1.2 (3H, d); 2.25(1H, m); 2.45 (1H, m); 2.6 (1H, m); 3.7 (2H, t); 4.2 (2H, q); 5.0 (1H,s); 5.05 (1H, d); 5.2 (2H, s); 5.6 (1H, d); 5.7 (1H, m); 5.8 (1H, d).

Hydroboration of the allyl group

To a flame-dried flask under flushing argon containing 0.74 g (2.4 mmol,1 eq) of the allylation product 11 dissolved in 20 mL of freshlydistilled THF and cooled to 0° C. in an ice/H₂O bath was added 1.25 mL(2.5 mmol, 1.05 eq) of BH₃.THF by syringe. The reaction was allowed tostir for two hours at which time TLC indicated the reaction wasfinished. The reaction was stopped by sequential addition of 8 mL ofmethanol, 3 mL of 5M NaOH and 3 mL of 30% H₂O₂. The solution wasacidified to pH 4 with 1% HCl and extracted with Et₂O (3×20 mL). Thecombined organic layers were dried with Na₂SO₄, filtered and evaporatedon a rotary evaporator. The residue was placed on a SiO₂ column andeluted with 2:1 ethyl acetate/ toluene. This yielded 0.30 g (38%) of aclear oil.

¹H NMR (300 MHz, CDCl₃): δ0.0 (9H, s); 0.9 (2H, t); 1.25 (3H, d);1.5-1.8 (4H, m); 2.55 (1H, m); 2.45 (1H, m); 2.6 (1H, m); 3.65 (2H, t);3.75 (2H, t); 5.2 (2H, s); 5.6 (1H, d); 5.8 (1H, d).

Swern oxidation of the alcohol to produce 12

A flame-dried flask under flushing argon containing 0.105 mL (1.5 mmol,2.55 eq) of DMSO dissolved in 5 mL of anhydrous CH₂Cl₂ was cooled to−78° C. in a dry ice/acetone bath. To this solution was added 0.058 mL(0.64 mmol, 1.14 eq) of oxalyl chloride by syringe. The solution wasallowed to stir for 5 minutes. 0.19 g (0.58 mmol, 1 eq) of the alcoholdissolved in 5 mL of anhydrous CH₂Cl₂ was added to the solution slowlyover the course of 5 minutes. The solution was allowed to stir for 30minutes. The reaction was terminated by the addition of 0.209 mL (1.5mmol, 2.6 eq) of triethylamine and stirring for 5 minutes beforeallowing the solution to warm to room temperature. The solution wasdiluted with 10 mL of H₂O and the aqueous and organic layers separated.The aqueous layer was extracted with CH₂Cl₂ (2×10 mL). The combinedorganic layers were dried with Na₂SO₄, filtered and evaporated on arotary evaporator. The residue was placed on a SiO₂ column and elutedwith 2:1 toluene/ethyl acetate. This yielded 0.15 g (80%) of 12 as aclear oil. ¹H NMR showed this compound identical to the aldehydeprepared using the 3-bromopropionaldehyde dimethyl acetal pathway.

Horner-Emmons-Wadsworth reaction of the aldehyde

To a flame-dried flask under flushing argon in an ice/H₂O bathcontaining 0.223 g (1.1 mmol, 1.05 eq) of the triethyl phosphonoacetatedissolved in 15 mL of freshly distilled THF was added 45 mg (1.1 mmol,1.05 eq) of NaH as a 60% mineral oil dispersion. Once hydrogen evolutionsubsided, the aldehyde dissolved in THF (3 mL) was added by syringe. Thesyringe was washed with an additional 2 mL of THF and the solution addedto the reaction. Almost immediately, an orange oil formed. Afterstirring overnight, the reaction mixture was quenched by the addition of20 mL of H₂O and the solution was made acidic with 1% HCl. The solutionwas extracted with Et₂O (3×10 mL). The combined organics were extractedwith saturated NaHCO₃ (1×10 mL) and brine (1×10 mL). The organic layerwas dried with Na₂SO₄, filtered and evaporated on a rotary evaporator.The residue was placed on a SiO₂ column and eluted with 2:1 hexane/ethylacetate. This yielded 0.39 g (92%) of a clear oil. ¹H NMR showed thiscompound identical to the HEW adduct prepared using the3-bromopropionaldehyde dimethyl acetal pathway.

Removal of the SEM protecting group to produce 10

In a round-bottomed flask was placed 0.55 g (1.4 mmol, 1 eq) of thepyrone and it was dissolved in 33 mL of a 10:1 mixture of THF/EtOH. Tothis solution was added 30 drops of concentrated H₂SO₄ and the reactionleft to stir. After 6 hours, TLC indicated no starting material. Thereaction was diluted with 30 mL of ethyl acetate. The solution wasextracted with saturated NaHCO₃ (3×20 mL). The aqueous layer wasacidified to <pH 4 with 10% HCl. The solution was extracted with ethylacetate (3×30 mL). The combined organic layers were dried with Na₂SO₄,filtered and evaporated on a rotary evaporator. This yielded 0.37 g(>98%) of 10 as a clear oil. The product needed no further purification.

¹H NMR (300 MHz, CDCl₃): δ1.25 (3H, d); 1.25 (3H, t); 1.7 (1H, m); 1.9(1H, m); 2.2 (2H, m); 2.6 (1H, m); 4.2 (2H, q); 5.5 (1H, d); 5.8 (1H,d); 5.95 (1H, d); 6.9 (1H, dt).

EXAMPLE 4 Synthesis of the 3-position side-chain Horner-Emmons-Wadsworthreaction of 2-pentanone with triethyl phosphonoacetate

To a flame-dried flask under flushing argon in an ice/H₂O bathcontaining 7.48 mL (37.7 mmol, 1 eq) of the triethyl phosphonoacetatedissolved in 100 mL of freshly distilled toluene was added 0.95 g (40mmol, 1.05 eq) of NaH as a 60% mineral oil dispersion. Once hydrogenevolution subsided, 2-pentanone was added. Almost immediately, an orangeoil formed. After stirring overnight, the reaction was quenched by theaddition of 50 mL of H₂O and the solution was made acidic with 1% HCland extracted with Et₂O (3×20 mL). The combined organics were extractedwith saturated NaHCO₃ (1×10 mL) and brine (1×10 mL). The organic layerwas dried with Na₂SO₄, filtered and evaporated on a rotary evaporator.The residue was placed on a SiO₂ column and eluted with 15:1hexane/ethyl acetate. This yielded 4.9 g (83%) of a clear oil.

¹H NMR (300 MHz, CDCl₃): δ0.9 (3H, t); 1.25 (3H, t); 1.5 (2H, m); 2.1(2H, t) 2.15 (3H, s); 4.15 (2H, q); 5.65 (1H, s).

Alternate synthesis of the unsaturated ester 16

To a flask containing 45.7 g (240 mmol, 1 eq) of Cul suspended in 240 mLof diethyl ether cooled to 0° C. with an ice/H₂O bath was added 343 mL(480 mmol, 2 eq) of a 1.4M solution of MeLi over the course of 1 hour.The solution was stirred for 5 minutes at 0° C. and then cooled to −78°C. with a dry ice/acetone bath. Initially, a yellow precipitate formedupon the addition of the MeLi but solubilized with time.

In a second flask, 18.98 g (120 mmol, 1 eq) of ethylbutyryl acetate in160 mL of diethyl ether was added to a suspension of 5.28 g (132 mmol,1.1 eq) of NaH in 80 mL of diethyl ether cooled to 0° C. over the courseof 1 hour. The slurry that formed was stirred for 20 minutes at 0° C.19.1 mL (132 mmol, 1.1. eq) of diethylphosphoro-chloridate was addedover the course of 10 minutes. The solution was stirred at roomtemperature for 2 hours and then poured into 200 mL of ice/saturatedNH₄Cl. The aqueous and organic layers were separated. The organic layerwas washed with 300 mL of saturated NaHCO₃. The organic layer was driedwith Na₂SO₄, filtered and evaporated on a rotary evaporator. Thisyielded the 35.4 g of the enol phosphonate which was used withoutfurther purification. The enol phosphonate was dissolved in 120 mL ofdiethyl ether and added to the first flask containing LiMe₂Cu over thecourse of 20 minutes with the solution was being cooled to −78° C. Thesolution was stirred for 2.5 hours while being cooled at −78° C. Thesolution was poured into 300 mL of saturated NH₄Cl. The aqueous andorganic layers were separated. The organic layer was washed with 2×200mL of saturated NaHCO₃ and 300 mL of brine solution. The organic layerwas dried with Na₂SO₄, filtered and evaporated on a rotary evaporator.This yielded 17 g of a light green liquid that was distilled to give10.4 g (55%) yield of a colorless oil. The NMR was identical to that of16 but contained a 10:1 ratio of E/Z isomers.

DIBAL reduction of the ethyl ester

A flame-dried flask under flushing argon containing 4.9 g (31.4 mmol, 1eq) of the ethyl ester dissolved in 100 mL of freshly distilled THF wascooled to −78° C. with a dry ice/acetone bath. 78.5 mL (78.5 mmol, 2.5eq) of a 1.0M solution of DIBAL was dripped into the solution by syringeover the course of 10 minutes. The solution was left to stir for 1 hour.Fifty milliliters of methanol were poured into the solution to quenchthe excess DIBAL. The solution was diluted with 200 mL of H₂O. 200 mL ofEt₂O was added to the solution followed by 100 mL of 5% HCl. The wholesolution was poured into a separatory funnel and the aqueous and organiclayers separated. The aqueous layer was extracted with Et₂O (2×50 mL).The combined organic layers were dried with Na₂SO₄, filtered andevaporated on a rotary evaporator. The residue was placed on a SiO₂column and eluted with 3:1 hexane/ethyl acetate. This yielded 1.2 g(34%) of a clear oil.

¹H NMR (300 MHz, CDCl₃): δ0.85 (3H, t); 1.4 (2H, m); 1.65 (3H, s); 1.9(2H, t); 1.4 (2H, d); 5.4 (1H, t).

Swern oxidation of the alcohol to produce 14

A flame-dried flask under flushing argon containing 1.9 mL (26.8 mmol,2.55 eq) of DMSO dissolved in 30 mL of anhydrous CH₂Cl₂ was cooled to−78° C. in a dry ice/acetone bath. To this solution was added 1.05 mL(12 mmol, 1.14 eq) of oxalyl chloride by syringe. The solution wasallowed to stir for 5 minutes. 1.2 g (10.5 mmol, 1 eq) of the alcoholdissolved in 10 mL of anhydrous CH₂Cl₂ was added to the solution slowlyover the course of 5 minutes. The syringe was washed with 10 mL ofCH₂Cl₂ and this solution added to the reaction. The solution was allowedto stir for 30 minutes. The reaction was stopped by the addition of 3.81mL (27.3 mmol, 2.6 eq) of triethylamine and stirring for 5 minutesbefore allowing the solution to warm to room temperature. The solutionwas diluted with 20 mL of H₂O and the aqueous and organic layersseparated. The aqueous layer was extracted with CH₂Cl₂ (2×10 mL). Thecombined organic layers were extracted with brine solution, dried withNa₂SO₄, filtered and evaporated on a rotary evaporator. The residue wasplaced on a SiO₂ column and eluted with 2:1 toluene/ethyl acetate. Thisyielded 0.45 g (38%) of a clear oil 14.

¹H NMR (300 MHz, CDCl₃): δ0.9 (3H, t); 1.55 (2H, m); 2.15 (3H, s); 2.2(2H, t); 5.9 (1H, d); 10.0 (1H, d).

Horner-Emmons-Wadsworth reaction of 14 with triethyl2-phosphonopropionate

To a flame-dried flask under flushing argon in an ice/H₂O bathcontaining 0.86 mL (4.0 mmol, 1 eq) of the triethyl2-phosphonopropionate dissolved in 100 mL of freshly distilled toluenewas added 0.10 g (4.2 mmol, 1.05 eq) of NaH as a 60% mineral oildispersion. Once hydrogen evolution subsided, the aldehyde 14 was added.Almost immediately, an orange oil came out of solution. The reaction wasleft to stir overnight. The reaction was stopped by the addition of 20mL of H₂O and the solution made acidic with 10% HCl. The solution wasextracted with Et₂O (3×20 mL). The combined organics were extracted withsaturated NaHCO₃ (1×10 mL) and brine (1×10 mL). The organic layer wasdried with Na₂SO₄, filtered and evaporated on a rotary evaporator. Theresidue was placed on a SiO₂ column and eluted with 11:1 hexane/ethylacetate. This yielded 0.385 g (50%) of a clear oil.

¹H NMR (300 MHz, CDCl₃): δ0.9 (3H, t); 1.3 (3H, t); 1.5 (2H, m); 1.85(3H, s); 1.95 (3H, s); 2.15 (2H, t) 4.15 (2H, q); 6.1 (1H, d); 7.5 (1H,d).

DIBAL reduction of the ethyl ester

A flame-dried flask under flushing argon containing 1.86 g (9.5 mmol, 1eq) of the ethyl ester dissolved in 40 mL of freshly distilled THF wascooled to −78° C. with a dry ice/acetone bath. 23.7 mL (23.7 mmol, 2.5eq) of a 1.0M solution of DIBAL was dripped into the solution by syringeover the course of 5 minutes. The solution was left to stir for 2 hours.An additional 5 mL of DIBAL was added. One hour later, 20 mL of methanolwas poured into the solution to quench the excess DIBAL. The solutionwas diluted with 50 mL of H₂O. Fifty milliliters of Et₂O were added tothe solution followed by 25 mL of 5% HCl. The whole solution was pouredinto a separatory funnel and the aqueous and organic layers separated.The aqueous layer was extracted with Et₂O (2×20 mL). The combinedorganic layers were dried with Na₂SO₄, filtered and evaporated on arotary evaporator. The residue was placed on a SiO₂ column and elutedwith 3:1 hexane/ethyl acetate. This yielded 1.32 g (91%) of a clear oil.

¹H NMR (300 MHz, CDCl₃): δ0.9 (3H, t); 1.45 (2H, m); 1.75 (3H, s); 1.8(3H, s); 2.05 (2H, t); 4.1 (2H, d); 6.0 (1H, d); 6.25 (1H, d).

DDQ oxidation of the alcohol to the aldehyde 1

To a flame-dried flask under flushing argon containing 0.7576 g (4.9mmol, 1 eq) of the alcohol dissolved in 25 mL of freshly distilled THFwas added 2.26 g (25 mmol, 2 eq) of DDQ. The reaction was allowed tostir for 2 hours. At this point, 5 g of SiO₂ were added and thevolatiles evaporated on a rotary evaporator. The resulting solid wasplaced on top of a SiO₂ column and the product eluted with 7:1hexanes/ethyl acetate. This yielded 0.39 g (52%) of 1 as a clear oil.

¹H NMR (300 MHz, CDCl₃): δ0.9 (3H, t); 1.55 (2H, m); 1.85 (3H, s); 1.95(3H, s); 2.2 (2H, t) 6.3 (1H, d); 7.1 (1H, d); 9.45 (1H, s).

EXAMPLE 5 Attachment of the 3-position side chain Acid-catalyzed aldolreaction between pyrone 10 and aldehyde

To a flamedried flask under flushing argon containing 0.172 g (650 μmol,1 eq) of the pyrone 10 dissolved in 20 mL of anhydrous CH₂Cl₂ was added34 μl (325 μmol, 0.5 eq) of TFA along with 1 g of 4 Å sieves. Thereaction was allowed to stir for 5 minutes. At this point, 100 μl (650μl, 1 eq) of the aldehyde 1 was added. The reaction was capped andheated at 45° C. for 24 hours. An additional equivalent of TFA was addedalong with 2 mL of CH₂Cl₂. The reaction was heated for an additional 12hours. The reaction was filtered. One gram of SiO₂ was added to thesolution and the solvent evaporated. The product became adsorbed ontothe SiO₂. The resulting solid was applied to the top of a SiO₂ columnand eluted successively with 7:1 hexane/ethyl acetate, 2:1 hexane/ethylacetate, 10:1 ethyl acetate/chloroform with 2 drops of acetic acid forevery 6 mL of eluent. This yielded 45 mg of the aldehyde, 1.5 mg of theproduct (1%) 13 and 75 mg of the pyrone.

¹H NMR (300 MHz, CDCl₃): δ0.85 (3H, t); 1.2 (3H, d); 1.25 (3H, t); 1.55(2H, m); 1.65 (1H, m); 1.7 (3H, s); 1.75 (3H, s); 1.9 (1H, m); 2.05 (2H,t); 2.15 (2H, t); 2.55 (1H, m); 4.15 (2H, q); 5.4 (1H, d); 5.5 (1H, d);5.75 (1H, s); 5.8 (1H, d); 6.2 (1H, s); 6.9 (1H, m).

Acylation of pyrone 10 with propionyl chloride

To a flame-dried flask under flushing argon containing 35 mg (130 μmol,1 eq) of the pyrone 10 dissolved in 1 mL of anhydrous TFA was added 23μl (260 μmol, 2 eq) of propionyl chloride. The reaction was heated to50° C. for 1 hour. An additional 4 equivalents of propionyl chloridewere added and the reaction was heated for 12 hours. An additional 4equivalents of propionyl chloride were added and the reaction was heatedfor 3 hours. The reaction was partitioned between H₂O and ethyl acetate.The aqueous layer was extracted again with ethyl acetate and the organiclayers were combined. The combined organic layers were dried withNa₂SO₄, filtered and evaporated on a rotary evaporator. The residue wasplaced on a SiO₂ column and eluted with 2:1 hexane/ethyl acetate. Thisyielded 21 mg (50%) of a light orange oil 15.

¹H NMR (300 MHz, CDCl₃): δ1.15 (3H, t); 1.25 (3H, d); 1.25 (3H, t); 1.7(1H, m); 1.9 (1H, m); 2.2 (2H, q); 2.6 (1H, m); 3.1 (2H, q); 4.2 (2H,q); 5.8 (1H, d); 5.95 (1H, s); 6.9 (1H, dt).

Base-catalyzed aldol reaction between pyrone and aldehyde to produce 17

To a flame-dried flask in a −78° C. dry ice/acetone bath under flushingargon containing 21 mg (65 μmol, 1 eq) of the pyrone 15 dissolved in 3mL of freshly distilled THF was added 2.9 mL (230 μmol, 3.6 eq) of 0.08MLDA by syringe. The reaction went from a light orange to a dark orange.The reaction was allowed to stir for 30 minutes. The aldehyde was addedby syringe. The color of the solution became just a little lighter.After stirring for 90 minutes, the reaction was quenched by addition ofsaturated NH₄Cl solution. Ten milliliters of ethyl acetate were added todilute the solution. 1% HCl solution was used to make the solutionacidic. The aqueous and organic layers were separated. The aqueous layerwas extracted with ethyl acetate (2×5 mL). The combined organic layerswere dried with Na₂SO₄, filtered and evaporated on a rotary evaporator.The residue was placed on a SiO₂ column and eluted with 4:4:1 hexane/ethyl acetate/ methanol. This yielded 2 mg (8%) of a light yellow oil17.

¹H NMR (300 MHz, CDCl₃): δ0.9 (3H, t); 1.25 (3H, d); 1.25 (3H, t); 1.5(2H, m); 1.7 (1H, m); 1.85 (3H, t); 1.9 (1H, m); 1.95 (3H, t); 2.15 (2H,t); 2.2 (2H, t); 2.6 (1H, m); 4.2 (2H, q); 5.8 (1H, d); 5.95 (1H, s);6.15 (1H, d); 6.9 (1H, dt); 7.0 (1H, d).

Hydrolysis of the ester 17 to the acid

To a round-bottomed flask containing 4.0 mg (10 μmol, 1 eq) of thepyrone ester 17 dissolved in 5 mL of 2:2:1 methanol/THF/H₂O is added 4mg (100 μmol, 10 eq) of LiOH.H₂O. The solution is stirred at roomtemperature for 6 hours. The volatiles are evaporated on the rotaryevaporator. Any remaining base is quenched by the addition of 1% HClsolution. The aqueous solution is extracted with ethyl acetate (3×10mL). The combined organic layers are dried with Na₂SO₄, filtered andevaporated on a rotary evaporator. 2 mg of an oily yellow residue isisolated and the residue is used in the next step without any furtherpurification.

¹H NMR (300 MHz, CDCl₃): δ0.9 (3H, t); 1.25 (3H, d); 1.5 (2H, m); 1.7(1H, m); 1.85 (3H, t); 1.9 (1H, m); 1.95 (3H, t); 2.15 (2H, t); 2.2 (2H,t); 2.6 (1H, m); 5.8 (1H, d); 5.95 (1H, s); 6.15 (1H, d); 6.95 (1H, dt);7.0 (1H, d).

Curtius sequence

To a flame-dried flask under flushing argon containing 2 mg (5.4 μmol, 1eq) of the residue from the hydrolysis dissolved in 1 mL of acetone isadded 1.8 μl (13 μmol, 2.4 eq) of DIPEA. The solution is cooled to 0° C.using an ice/H₂O bath. A solution of 0.83 μl (11 μmol, 2 eq) of methylchloroformate dissolved in acetone is added dropwise over 30 minutes.After the reaction stirs for 30 minutes, a solution of 1.4 mg (2.2 mmol,4.0 eq) of NaN₃ dissolved in H₂O was added. The solution is stirred for15 minutes and then poured into 30 mL of ice H₂O. The acyl azide isextracted with 6-2 mL portions of toluene. The combined organic layersare dried with Na₂SO₄, filtered and concentrated to 2 mL on a rotaryevaporator. This solution is added slowly over the course of 15 minutesto a vigorously refluxing solution of 250 μl of methanol in 1 mL drytoluene. Reflux is maintained for 30 minutes and the volatiles areevaporated on a rotary evaporator. The residue is placed on a SiO₂ prepplate and eluted with 95:5 CH₂Cl₂/methanol. This yields 0.5 mg of 18 asa light yellow oil.

¹H NMR (300 MHz, CDCl₃): δ0.9 (3H, t); 1.25 (3H, d); 1.5 (2H, m); 1.7(1H, m); 1.85 (3H, t); 1.9 (1H, m); 1.95 (3H, t); 2.15 (2H, t); 2.2 (2H,t); 2.6 (1H, m); 3.7 (3H, s); 4.9 (1H, m); 5.95 (1H, s); 6.15 (1H, d);6.4 (1H, m); 7.0 (1H, d).

EXAMPLE 6 Modified Curtius Rearrangement

Compound 17 a (35 mg, 0.09 mmol), diphenylphosphoryl azide (124 mg, 0.45mmol) and Et₃N (63 μL, 0.45 mmol) was dissolved in C₆H₆ (8.0 mL) andrefluxed for 9.0 h. The reaction mixture was cooled to RT, and anhydrousMeOH (2.0 mL) was added. The reaction mixture was warmed to reflux for6.0 h, and then concentrated in vacuo. The remaining organic residue wasflash chromatographed (30% EtOAc in hexanes as eluant) to provide(±)-myxopyronin A (26.6 mg, 71% yield) as a yellow oil.

¹H NMR (300 MHz, CDCl₃): δ7.0 (d,J=11.6 Hz, 1H); 6.47 (m,1H); 6.23 (m,1H); 6.16 (d,J=11.5 Hz, 1H); 5.94 (s, 1H); 4.94 (m, 1H); 3.72 (s, 3H);2.6 (m, 1H); 2.15 (t,j=7.7 Hz, 2H); 2.05 (m, 2H); 2.01 (s, 3H); 1.85 (s,3H); 1.76 (m, 1H); 1.57 (m, 1H); 1.51 (m, 1H); 1.25 (d,J=6.8 Hz, 3H);0.92 (t,J=7.3 Hz, 3H). ¹H NMR (300 MHz, CD₃OD): δ7.15 (d,j=12.0 Hz, 1H);6.39 (d,j=14.2 Hz, 1H); 6.27 (d,j=12.0 Hz, 1H); 6.10 (s, 1H); 5.03(dt,J=7.1, 14.0 Hz, 1H); 3.66 (s, 3H); 2.68 (m, 1H); 2.19 (t,J=7.4 Hz,2H); 2.01 (m, 2H); 1.93 (s, 3H); 1.81 (s, 3H); 1.76 (m,1H); 1.59 (m,1H);1.52 (m, 2H); 1.25 (d,j=6.9 Hz, 3H); 0.92 (t,j=7.2 Hz, 3H). MS (FABMS):(M+H) calc. 418, anal. 418. IR (Film, KBr): ν_(max)=3318 m (b), 2966 s,2931 s, 2872 m, 1736 s,1719 s, 1684 m, 1637 s, 1560 s, 1542 s, 1525 s,1437 m, 1378 m, 1237 m, 1049 m. UV (methanol): λ_(max) (log ε): 224 nm(4.17), 295 nm (4.04).

Saponification of pyrone ester 17

To a round-bottomed flask containing 25.8 mg (62 μmol, 1 equiv) of thepyrone ester 17 dissolved in 5 mL of 2:2:1 methanol/THF/H₂O was added 26mg (620 μmol, 10 equiv) of LiOH H₂O. The solution is stirred at RT for24 h. The reaction was quenched by the addition of 1% HCl solution. Theaqueous solution was extracted with ethyl acetate (3×10 mL). Thecombined organic layers were dried with Na₂SO₄, filtered and evaporatedin vacuo to provide 25 mg of an oily yellow residue. The residue(compound 17 a) was isolated and used in the next step without furtherpurification.

¹H NMR (400 MHz, CDCl₃): δ7.03 (dt,J=6.9,15.7 Hz, 1H); 7.00 (d,j=11.7Hz, 1H); 6.16 (d,J=11.6 Hz, 1H); 5.96 (s, 1H); 5.85 (d,J=15.7 Hz, 1H);2.61 (m, 1H); 2.25 (m, 2H); 2.15 (t,j=7.6 Hz, 2H); 2.00 (s, 3H); 1.91(m, 1H); 1.85 (s, 3H); 1.69 (m, 1H); 1.49 (m, 2H ); 1.27 (d,J=6.9 Hz,3H); 0.91 (t,J=7.3 Hz, 3H). MS (FABMS): (M+H) calc. 389, anal. 389. IR(Film, KBr): ν_(max)=3436 m (b), 2966 s,2931 s,2872 m, 1731 s, 1713 s,1613 s, 1548 s (b), 1443 m, 1384 m, 1255 m.

Preparation of allyl alcohol 14

To a solution of freshly distilled COCl₂ (480 μL, 5.5 mmol) in CH₂Cl₂(15 mL) was added DMSO (850 μL, 12 mmol) at −78° C. The reaction mixturewas stirred at −78 ° C. for 30 min, then the starting alcohol 13 a (570mg, 5.0 mmol, dissolved in 3.0 mL CH₂Cl₂, plus 2.0 mL wash) was added.This whole reaction mixture was stirred at −78° C. for another 30 minthen Et₃N (3.5 mL, 25 mmol) was added. The reaction was stirred for 1.0h at −78 ° C., then warmed to RT for 1.0 h before being quenched by H₂O.The reaction mixture was extracted with CH₂Cl₂ (2×80 mL), dried (MgSO₄),filtered and concentrated in vacuo to afford the crude aldehyde. Thecrude product was purified by flash chromatography (10% EtOAc in hexanesas eluant) to provide viscous aldehyde 14 (548 mg, 98% yield) as acolorless oil.

¹H NMR (400 MHz, CDCl₃): δ0.91 (t, 3H); 1.51 (m, 2H); 2.14 (s, 3H); 2.2(t, 2H); 5.9 (d, 1H); 10.0 (d, 1H). ¹³C NMR (67.5 MHz, CDCl₃): δ14.01;17.81; 20.69; 42.96; 127.75; 164.49; 191.67.

Preparation of pyrone 5

To a flame-dried flask under a fluching argon atmosphere containingcarboxylic acid 4 a (10.55 g, 91 mmol, 1 equiv dissolved in 250 mL offreshly distilled THF) was added carbonyidiimidazole (16.22 g, 100 mmol,1.1 equiv). The reaction was left stirring for 12 h before beingconcentrated on a rotary evaporator. The residue was partitioned between100 mL of CHCl₃ and 100 mL of 10% HCl. The aqueous layer was extractedwith CHCl₃ (2×50 mL). The combined organic layers were dried withNa₂SO₄, filtered and evaporated on a rotary evaporator. The productneeded no further purification provided it is left on a vacuum pump forsufficient duration to remove any remaining starting acid. This yieldedcompound 5 (8.8 g, 98%) as a tan solid.

¹H NMR (300 MHz, CDCl₃): δ5.91 (s, 1H); 3.10 (q,j=7.3 Hz, 2H); 2.53 (q,j=7.2 Hz, 2H); 1.24 (t,j=7.5 Hz, 3H); 1.15 (t,J=7.2 Hz, 3H). ¹³C NMR (75MHz, CDCl₃): δ208.36; 181.16; 173.52; 161.16; 99.92; 99.56; 35.372;27.52; 10.49; 7.78. MS (El): calc. 196.0, anal. 196.0. IR (Film, KBr):ν_(max)=3400 m (b), 3020 s, 1710 s (b), 1625 m (b), 1555 s (b), 20 1430w (b), 1315 vs, 760 vs (b).

Preparation iodide 8 b

To a solution of imidazole (10.2 g, 150 mmol) and triphenylphosphine(14.4 g, 55 mmol) in CH₂Cl₂ (200 mL) at 0° C. was added 12 (14.0 g, 55mmol). After 10 min, a solution of alcohol 8 a (9.5 g, 50 mmol) inCH₂Cl₂ (100 mL) was added over 5 min. The mixture was warmed to RT,covered in aluminum foil, and stirred for an additional 15 h in thedark. The reaction was then diluted with 2.0 mL saturated Na₂S₂O₄aqueous solution before further dilution with water (150 mL). Theorganic layer was separated and the aqueous layer was back extractedwith CH₂Cl₂ (2×80 mL). The combined organic layers were dried (MgSO₄),filtered, and concentrated in vacuo. The crude product was purified byflash chromatography (2% EtOAc in hexanes as eluant) to afford iodide 8b (14.4 g, 96% yield) as a pale oil.

¹H NMR (400 MHz, CDCl₃): δ3.64 (t, 2H); 3.25 (t, 2H); 1.96 (m, 2H); 0.87(s, 9H); 0.05 (s, 6H). ¹³C NMR (67.5 MHz, CDCl₃): δ62.32; 36.14; 25.90;8.26; 3.61; −5.33.

Preparation of allyl alcohol 13 a

To a white slurry solution of zirconocene dichloride (11.7 g, 40 mmol)in 100 mL (CH₂)₂Cl₂ was added AlMe₃ (40 mL, 2.0 M in hexanes, 80 mmol)at 0° C., stirred for 45 min, and then warmed to RT for 1.5 h. To thislemon-yellow solution was added 1-pentyne (2.72 g, 40 mmol, dissolved in20 mL (CH₂)₂Cl₂) at RT. The reaction was allowed to stir for 3.0 h. Thenthe solvent and the unreacted trimethylalane were evaporated underreduced pressure (maximum 50° C., 0.3 mm Hg, 2˜3 h). The remainingorange-yellow organic residue was extracted with dry hexanes (4×30 mL),and the yellow extract was transferred to a 500 mL round-bottom flaskvia a cannula. To this was added n-BuLi (16 mL, 2.5 M in hexanes, 40mmol) at 0° C. This orange-yellow slurry/solution was stirred from 0° C.to RT for 1.5 h, and then THF (70 mL) was added to dissolve theprecipitate. The resulting solution (homogeneous, brown-yellow color)was cannulated to a suspension of paraformaldehyde in THF under a N₂atmosphere. This orange-yellow suspension solution was allowed to stirat RT for 20 min.

The reaction was cooled to 0° C. (ice water bath), ice was added toquench the reaction, and then saturated NH₄Cl (100 mL) was added. Theice bath was removed and the reaction was further acidified with 3 M HCluntil the reaction turned to a clear yellow (homogeneous) solution. Atthis time, the reaction pH was 2˜3. The organic layer was separated, andthe aqueous layer was extracted with ether (2×150 mL). The organicextracts were combined and washed with a saturated solution NaHCO₃ (200mL), then dried with Na₂SO₄, filtered and concentrated under reducedpressure to provide crude allylic alcohol 13 a. The crude product waspurified by flash chromatography (20% EtOAc in hexanes as eluant) toafford alcohol 13 a (3.37 g, 74% yield) as a colorless oil.

¹H NMR (400 MHz, CDCl₃): δ5.4 (t, 1H); 4.1 (d, 2H); 1.9 (t, 2H); 1.65(s, 3H); 1.4 (m, 2H); 0.85 (t, 3H). ¹³C NMR (75 MHz, CDCl₃): δ139.15;123.33; 59.01; 58.97; 41.54; 41.50; 20.61; 15.88; 13.57.

Preparation of acid 4 a

Ethyl propionate (25 g, 175 mmol) was dissolved in 30 mL of 1.5 Msolution NaOH and stirred at RT for 38 h. The reaction was cooled to 0°C., and 3 M HCl was slowly added until the reaction system reached apH˜1, then solid KCl was added to saturate the reaction, followed byEtOAc extraction (3×100 mL), and CHCl₃ (2×100 mL). The combined organiclayers were dried with Na₂SO₄, filtered and evaporated on a rotaryevaporator. This protocol afforded the carboxylic acid 4 a (15.5 g, 76%yield) as a white solid. The product was used in the next step withoutfurther purification.

¹H NMR (300 MHz, CDCl₃): δ3.52 (s, 2H); 2.60 (q,J=7.3 Hz, 2H); 1.10(t,J=7.3 Hz, 3H). ^(—)C NMR (75 MHz, CDCl₃): δ204.28; 172.32; 47.87;36.60; 7.45. MS (El): calc. 116.1, anal. 116.1. IR (Film, KBr):ν_(max)=3400 m (b), 3020 s, 1708 s (b), 1620 w, 1410 m, 1300 m, 1218 vs,1110 w, 1040 w, 925 w, 760 vs.

Preparation of alcohol 8 a

To a suspension of NaH (4.0 g, 100 mmol, 60% dispension in mineral oil)in THF (100 mL) at RT was added 1,3-propane diol (7.6 g, 100 mmol,dissolved in 50 mL of THF) via a cannula. The resulting mixture wasstirred at RT for 45 min, then a solution of TBSCl (15.0 g, 100 mmol) in50 mL THF was added to the reaction mixture by cannula. This resultingmixture was stirred for 1.0 h at RT. The reaction was quenched withsaturated NaHCO₃ aqueous solution (200 mL), and extracted with ether(2×200 mL). The combined organic layers were dried (MgSO₄), filtered,and concentrated in vacuo. The crude product was purified by flashchromatography (20% EtOAc in petroleum ether as eluant) to afford thepure mono-protected alcohol 8 a (18.1 g, 98% yield) as a clear oil.

¹H NMR (400 MHz, CDCl₃): δ3.73-3.80 (m, 4H); 2.71 (br s, 1H); 1.74 (m,2H); 0,86 (s, 9H); 0.04 (s, 6H). ¹³C NMR (75 MHz, CDCl₃): δ62.70; 62.16;34.22; 25.89; 25.81; 25.76; 18.13; −5.55.

Preparation of pyrone 5 a

To a stirred solution of diisopropylamine (3.34 mL, 24 mmol) in 25 mLTHF at −78° C. under an argon atmosphere was added n-butyl lithium (9.45mL, 2.5 M solution in hexanes, 23.6 mmol). The mixture was allowed towarm to 0° C. for 30 min and then recooled to −78° C. The resultingsolution was treated with a solution of pyrone 5 (1.47 g, 7.5 mmol) in10 mL of THF, and stirred for 1.0 h at −78° C. The derived dianion wastreated with iodide 8 b (2.5 g, 8.25 mmol) in 10 mL THF, followed by theaddition of HMPA (4.0 mL, 23.2 mmol). The reaction mixture was allowedto stir for 30 min at −78° C., before being diluted with saturated NH₄Claqueous solution (50 mL). The organic layer was separated, and theaqueous layer was extracted with ether (2×50 mL). All organic layerswere combined and dried (MgSO₄), filtered, and concentrated in vacuo.The resulting crude oil was purified by flash chromatography (20% EtOAcin hexanes as eluant) to afford the pure alkylation product 5 a (2.4 g,87% yield) as a yellow oil.

¹H NMR (400 MHz, CDCl₃): δ5.90 (s, 1H); 3.57 (t, 2H); 3.08 (q, 2H); 2.56(m, 1H); 1.43-1.73 (m, 3H); 1.22 (d,j=7.3 Hz, 3H); 1.13 (t, 3H); 0.86(s, 9H); 0.01 (s, 6H). ^(—)C NMR (67.5 MHz, CDCl₃): δ207.81; 180.57;175.56; 160.64; 99.28; 62.16; 38.27; 34.81; 29.99; 29.72; 25.44; 17.82;17.48; 7.27; −5.82.

Preparation of diol 5 b

A solution of TBS ether 5 a (1.66 g, 4.5 mmol) in 50 mL of AcOH, THF,and water (3:1:1) was stirred at RT for 15 h. The reaction was dilutedwith 50 mL water, extracted with CHCl₃ (2×100 mL). The combined organiclayers were washed with a NaHCO₃ saturated solution (1×200 mL). Theorganic layer was separated, and the aqueous layer was back extractedwith CHCl₃ (1×10 mL). All combined organic layers were dried (MgSO₄),filtered, and concentrated in vacuo. The crude product was purified byflash chromatography (40% EtOAc in hexanes as eluant) to afford diol 5 b(1.04 g, 91% yield) as a yellow oil.

¹H NMR (400 MHz, CDCl₃): δ5.90 (s, 1H); 3.63 (br s, 1H); 3.08 (q, 2H);2.58 (m, 1H); 1.71-1.80 (m, 1H); 1.50-1.62 (m, 3H); 1.33 (br s, 1H);1.23 (d,j=7.33 Hz, 3H); 1.13 (t, 3H). ¹³C NMR (67.5 MHz, CDCl₃):δ180.45; 175.20; 160.62; 99.30; 99.03; 61.62; 38.14; 34.71; 29.83;29.46; 17.32; 7.14; 1.51.

Preparation of aldehyde 5 c

To a solution of alcohol 5 b (483 mg, 1.9 mmol) in CH₂Cl₂ (20 mL) wasadded pyridine (845 μL, 10.45 mmol), followed by Dess-Martin periodinatereagent (2.8 mg, 6.65 mmol) in one portion. The resulting reactionmixture was stirred at RT for 2.0 h before being quenched with saturatedNaHCO₃ aqueous solution. The reaction mixture was extracted with CH₂Cl₂(2×60 mL), dried (MgSO₄), filtered, and concentrated in vacuo. The crudeproduct was flash chromatographed (15% EtOAc in hexanes as eluant) toafford aldehyde 5 c (417 mg, 87% yield) as yellow oil.

¹H NMR (400 MHz, CDCl₃): δ5.90 (s, 1H); 3.07 (q, 2H); 2.59 (m, 1H); 2.46(t, 2H); 1.83-2.02 (m, 2H); 1.23 (d,j=7.33 Hz, 3H); 1.12 (t, 3H). ¹³CNMR (67.5 MHz, CDCl₃): δ200.73; 180.69; 174.35; 160.62; 99.93; 99.45;40.82; 37.82; 35.00; 25.87; 17.52; 7.43; 1.81.

Preparation of acyl pyrone ester 15

To a solution of triethyl phosphonoacetate (1.4 g, 6.24 mmol) in C₆H₆(20 mL) at room temperature was added NaH (210 mg, 5.2 mmol, 60%dispersion in mineral oil). The resulting reaction mixture was stirredat RT for 15 min, before aldehyde 5 c (525 mg, 2.08 mmol) in 15 mL C₆H₆was added via cannula. The reaction was allowed to stir at RT for 1 hbefore being diluted with aqueous NH₄Cl solution (50 mL). The mixturewas extracted with EtOAc (2×100 mL), dried (MgSO₄), filtered, andconcentrated in vacuo. The crude product was flash chromatographed (15%EtOAc in hexanes as eluant) to afford compound 15 (596 mg, 89% yield) asa yellow oil.

¹H NMR (400 MHz, CDCl₃): δ6.89 (dt,J=6.8, 15.6 Hz, 1H); 5.93 (s, 1H);5.82 (d,J=15.6 Hz, 1H); 4.17 (q,j−7.1Hz, 2H); 3.11 (q,J=7.1Hz, 2H); 2.60(m, 1H); 2.21 (ddd,J=7.2, 7.3, 7.4 Hz, 2H); 1.89 (m, 1H); 1.69 (m, 1H);1.28 (t,J=7.1 Hz, 3H); 1.25 (d,J=6.9 Hz, 3H);1.16 (t,j=7.1Hz, 3H). ¹³CNMR (75 MHz, CDCl₃): δ208.38; 180.98; 174.93; 166.37; 161.00; 147.16;122.38; 100.17; 99.78; 60.37; 38.36; 35.40; 32.32; 29.59; 17.86; 14.29;7.77. IR (film, KBr): ν_(max)=2978 s, 2942 s, 1731 s (b), 1637 s, 1631s, 1554 s, 1443s,1272 m, 1178 m, 1043 m.

Preparation of pyrone diol 14 a

Compound 15 (55 mg, 0.17 mmol) was dissolved in 3.5 mL CH₂Cl₂ andstirred at −78° C. under argon atmosphere. To this yellow homogeneoussolution was added freshly distilled TiCl₄ (75 μl, 0.68 mmol); thereaction mixture became an orange-yellow slurry instantly. Afterstirring for 30 min at −78 ° C., Et₃N (104 μL, 0.75 mmol) was added, andthe reaction mixture became dark red. After stirring at −78° C. for 3.0h, aldehyde 14 (57 mg, 0.51 mmol) dissolved in 2.0 mL CH₂Cl₂ was addedvia a cannula. This dark red mixture was stirred at −78° C. for 17 hbefore it was quenched with distilled water, and extracted with CHCl₃(3×30 mL). The organic layers were dried (Na₂SO₄), and concentrated invacuo. The crude product was flash chromatographed (25% EtOAc in hexanesas eluant) to provide diol 14 a (53 mg, 71% yield) as a sticky yellowoil.

¹H NMR (400 MHz, CDCl₃): δ6.86 (dt, 1H); 5.90 (s, 1H); 5.80 (d,J=15.87Hz, 1H); 5.21 (d,j=8.55 Hz, 1H); 4.74 (m, 1H); 4.08-1.18 (m, 3H); 2.57(m, 1H); 2.42 (br s, 1H); 2.19 (dt, 2H); 1.94 (t, 2H); 1.83-1.90 (m,1H); 1.65 (s, 3H); 1.60-1.70 (m, 1H); 1.30-1,43 (m, 2H); 1.19-1.27 (m,9H); 0.82 (t, 3H).

Preparation of pyrone ester 17

A stirred solution of diol 14 a (52 mg, 0.12 mmol) in 4 mL CH₂Cl₂ wascooled to −15° C.

To this solution was added Et₃N (67 μL, 0.48 mmol), followed by MsCl (28μL, 0.36 mmol). The yellow solution was stirred at −15° C. for 30 min,and then warmed to 0° C. over 15 h. To this was added DBU (108 μL, 0.72mmol), the resulting reaction mixture was stirred from 0° C. to RT overa 12 h time period before being diluted with 2% HCl aqueous solution (10mL). The mixture was stirred for 5 min then extracted with EtOAc (2×15mL), dried (Na₂SO₄), filtered and concentrated in vacuo. The crudeproduct was flash chromatographed (20% EtOAc in hexanes as eluant) toprovide compound 17 (36 mg, 72% yield) as yellow oil.

¹H NMR (400 MHz, CDCl₃): δ7.00 (d,j=11.6 Hz, 1H); 6.91 (dt,J=6.8, 15.6Hz, 1H); 6.16 (d,J=11.6 Hz,1H); 5.95 (s,1H); 5.83 (d,J=15.6 Hz, 1H);4.19 (q,J=7.1 Hz, 2H); 2.61 (m, 1H); 2.24 (m, 2H); 2.15 (t,J=7.6 Hz,2H); 1.91 (m, 1H); 2.01 (s, 3H); 1.85 (s, 3H); 1.69 (m, 1H); 1.49 (m,2H); 1.29 (t,j=7.1 Hz, 3 H); 1.27 (d,J=6.9 Hz, 3H); 0.91 (t,J=7.3 Hz,3H). ¹³C NMR (75 MHz, CDCl₃): δ201.72; 180.83; 174.82; 166.42; 160.39;149.17; 147.25; 133.65; 133.04; 122.37; 120.67; 100.06; 99.17; 60.38;42.93; 38.27; 32.25; 29.62; 21.04; 17.81; 17.30; 14.29; 13.88; 13.55. MS(FABMS): (M+H) calc. 417, anal. 417. IR (Film, KBr): ν_(max)=2966 m,2931 m, 2872 m, 1748 s, 1736 s, 1719 s, 1701 s, 1560 s, 1542 s, 1454 s.

EXAMPLE 7 Preparation of Compound 15 a

3-(1-Propionyl)-4-hydroxy-6-ethyl-2-pyrone (5). Ethyl propionate (5.0 g,35 mmol) was dissolved in 30 mL of 1.5 M solution NaOH and stirred at RTfor 30 h. The reaction mixture was cooled to 0° C., and 3 M HCl wasslowly added until the mixture reached a pH of about 1; then solid KClwas added to saturate the solution. The reaction mixture was extractedwith EtOAc (3×100 mL), and CHCl₃ (2×100 mL), and the combined organiclayers were dried with Na₂SO₄, filtered and concentrated in vacuo toafforded the crude ethyl propionic acid (3.1 gm, 76% yield) as a whitesolid. The material was used without further purification in the nextstep:

¹H NMR (300 MHz, CDCl₃) δ3.52 (s, 2H), 2.60 (1,j=7.3 Hz, 2H), 1.10(t,J=7.3 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ204,3, 172.3, 47.9, 36.6,7.5; IR (neat) χ max: 3400, 3020, 1708, 1620, 1410, 1300,1218,1110,1040,925,760.

To a stirring solution of the above ethyl propionic acid (3.1 g, 26.7mmol) in 50 mL THF was added carbonyldiimidazole (5.6 gm, 34.7 mmol).The reaction was left stirring for 18 h before being quenched with 2%HCl aq. solution (20 mL, pH 2 to 3), and was extracted with EtOAc (3×50mL). The organic layers were dried (Na₂SO₄), filtered and thenconcentrated in vacuo. The product needed no further purification aslong as it was left on a vacuum pump for sufficient duration to removeany remaining starting acid. This protocol affords compound 5 (2.22 g,86%) as a pale yellow solid:

¹H NMR (400 MHz, CDCl₃) δ5.91 (s, 1H); 3.10 (1,j=7.3 Hz, 2H); 2.53 (q,J=7.2 Hz, 2H); 1.24 (t,J=7.5 Hz, 3H); 1.15 (t,J=7.2 Hz, 3H). ¹³C NMR (75MHz, CDCl₃) δ208.4, 181.2, 173.5, 161.2, 99.9, 99.6; 35.4, 27.5, 10.5,7.8; IR (neat)δ max: 3420, 2980,1731,1640,1437, 1014, 760 cm⁻¹.

1-lodo-3-tert-butyidimethylsilyloxy)propane (8 c). To a suspension ofNaH (4.0 g, 100 mmol, 60% dispersion in mineral oil) in THF (100 mL) atRT was added 1,3-propanediol (7.6 g, 100 mmol, dissolved in 50 mL ofTHF) via a cannula. The resulting mixture was stirred at RT for 45 min,before a solution of TBSCl (15.0 g, 100 mmol) in 50 mL THF was added bycannula. This resulting reaction was stirred for 1.0 h at RT. Thereaction was quenched with saturated aqueous NaHCO₃ solution (200 mL),and extracted with ether (2×200 mL). The combined organic layers weredried (MgSO₄), filtered, and concentrated in vacuo. The crude productwas purified by flash chromatography (20% EtOAc in petroleum ether aseluant) to afford the pure mono-protected alcohol (18.1 g, 98% yield) asa clear oil:

¹H NMR (400 MHz, CDCl₃) δ3.73-3.80 (m, 4H); 2.71 (br s, 1H); 1.74 (m,2H); 0.86 (s, 9H); 0.04 (s, 6H); ¹³C NMR (67.5 MHz, CDCl₃) δ62.7, 62.2,34.2, 25.9, 25.8,25.8, 18.1, −5.6; IR (neat)δ max: 3371, 2929, 2853,1473,1389, 1361, 1257, 1099, 1006, 837, 776, 662 cm⁻¹.

To a solution of imidazole (10.2 g, 150 mmol) and triphenylphosphine(14.4 gm, 55 mmol) in CH₂Cl₂ (200 mL) at 0° C. was added 12 (14.0 g, 55mmol). After 10 min solution of the above mono-protected alcohol (9.5 g,50 mmol) in CH₂Cl₂ (100 mL) was added over 5 min. The mixture was warmedto RT, covered in aluminum foil, and stirred for an additional 15 h inthe dark. The reaction was then diluted with 2.0 mL saturated Na₂S₂O₄aqueous solution before further dilution with water (150 mL). Theorganic layer was separated and the aqueous layer was back extractedwith CH₂Cl₂ (2×80 mL). The combined organic layers were dried (MgSO₄),filtered, and concentrated in vacuo. The crude product was purified byflash chromatography (2% EtOAc in hexanes as eluant) to afford theprimary iodide 8 c (14.4 gm, 96% yield) as a pale yellow oil:

¹H NMR (400 MHz, CDCl₃): δ3.64 (t,j=5.7 Hz, 2H), 3.25 (t,j=6.6 Hz, 2H),1.96 (m, 2H),1.96 (m, 2H), 0.87 (s, 9H), 0.05 (s, 6H); ¹³C NMR (67.5MHz, CDCl₃) δ62.3, 36.1, 25.9, 8.3, 3.6, −5.3; IR (neat)δ max: 2929,2857, 1472, 1101, 835, 776 cm⁻¹.

3-(1-Propionyl)4hydroxy-6-[4-methyl-1-(tert-butyldimethylsilyloxy)butane]-2-pyrone(5 a). To a stirred solution of diisopropylamine (3.34 mL, 24 mmol) in25 mL THF at −78° C. under argon was added n-butyl lithium (9.45 mL, 2.5M solution in hexanes, 23.6 mmol). The mixture was allowed to warm to 0°C. for 30 min, recooled to −78 ° C., and then treated with a solution ofpyrone 5 (1.47 gm, 7.5 mmol) in 10 mL of THF. After stirring for 1.0 hat −78° C., the derived dianion was treated with iodide 8 c (2.5 gm,8.25 mmol) in 10 mL THF, followed by the addition of HMPA (4.0 mL, 23.2mmol). The reaction mixture was allowed to stir for 30 min at −78° C.,before being diluted with saturated NH₄Cl aqueous solution (50 mL). Theorganic layer was separated, and the aqueous layer was extracted withether (2×50 mL). The organic layers were combined and dried (MgSO₄),filtered, and concentrated in vacuo. The resulting crude oil waspurified by flash chromatography (20% EtOAc in hexanes as eluant) toafford the pure alkylation product 5 a (2.4 gm, 87% yield) as a yellowoil:

¹H NMR (400 MHz, CDCl₃) δ5.90 (s, 1H), 3.57 (t,j=6.0 Hz, 2H), 3.08(q,J=7.3 Hz, 2H), 2.56 (m, 1H), 1.43-1.73 (m, 4H), 1.22 (d,J=7.3 Hz,3H), 1.13 (t, 3H), 0.86 (s, 9H), 0.01 (s, 6H); ^(—)C NMR (67.5 MHz,CDCl₃) δ207.8, 180.6, 175.6, 160.6, 99.3, 62.2, 38.3, 34.8, 30.0, 29.7,25.4, 17.8,17.5, 7.3, −5.8; IR (neat)δ max: 2936, 2858, 1743, 1636,1561, 1445, 1256, 1100, 1006, 835, 776, cm⁻¹; CIHRMS (NH₃ gas) calcd forC₁₉H₃₃SiO₆ (M+H⁺) 369.2085.

3-(1-Propionyl)-4-hydroxy-6-(4-methyl-butan-1-ol)-2-pyrone. A solutionof TBS ether 5 a (1.66 gm, 4.5 mmol) in 40 mL of AcOH, THF, and water(3:1:1) was stirred at RT for 22 h. The reaction was diluted with 50 mLwater, extracted with EtOAc (2×100 mL). The combined organic layers werewashed with NaHCO₃ saturated solution (1×100 mL). The organic layer wasseparated, and the aqueous layer was back extracted with EtOAc (1×100mL). The organic layer was separated, and the aqueous layer was backextracted with EtOAc (1×100 mL). The combined organic layers were dried(MgSO₄), filtered, and concentrated in vacuo. The crude product waspurified by flash chromatography (40% EtOAC in hexanes as eluant) toafford dialcohol (1.04 gm, 91% yield) as a yellow oil:

¹H NMR (400 MHz, CDCl₃) δ5.90 (s, 1H), 3.63 (br t, 2H), 3.08 (q, j=7.3Hz, 2H), 2.58 (m, 1H), 1.75 (m, 1H), 1.50-1.62 (m, 3H), 1.33 (br s, 1H),1.23 (d,j=7.3 Hz, 3H), 1.13 (t,j=7.3 Hz, 3H); ¹³C NMR (67.5 MHz, CDCl₃)δ180.5, 175.2, 160.6, 99.3, 99.0, 61.6, 38.1, 34.7, 29.8, 29.5, 17.3,7.1, 1.51; IR (neat)δ max: 3425, 2940, 1734, 1636, 1559, 1448, 1063,1014 cm⁻¹; CIHRMS (NH₃ gas) calcd for C₁₃H₁₉O₅ (M+H⁺) 255.1232, found:255.1252.

3-(1-Propionyl)-4-hydroxy-6-(4-methyl-butanol)-2pyrone (5 c). To asolution of dialcohol (483 mg, 1.9 mmol) in CH₂Cl₂ (20 mL) was addedpyridine (845 μL, 10.45 mmol), followed with Dess-Martin periodinatereagent (2.8 mg, 6.65 mmol) in one portion. The resulting reactionmixture was stirred at RT for 2.0 h before being quenched with saturatedNaHCO₃ aqueous solution. The reaction mixture was extracted with CH₂Cl₂(2×60 mL), dried (MgSO₄), filtered, and concentrated in vacuo. The crudeproduct was flash chromatographed (15% EtOAc in hexanes as eluant) toafford aldehyde 5 c (426 mg, 89% yield) as yellow oil:

¹H NMR (400 MHz, CDCl₃) δ9.73 (s, 1H), 5.90 (s, 1H), 3.07 (q,j=7.3 Hz,2H), 2.59 (m, 1H), 2.46 (t,j=7.3 Hz, 2H), 1.83-202 (m, 2H), 1.23(d,J=7.3 Hz, 3H), 1.12 (t,J=7.3 Hz, 3H); ¹³C NMR (67.5 MHz, CDCl₃)δ200.7, 180.7, 174.4, 160.6, 99.9, 99.5, 40.8, 37.8, 350, 25.9, 17.5,7.4, 1.8; IR (neat)δ max: 3096, 2978, 1727, 1636, 1560, 1446, 1391,1235, 1070, 1014, 832 cm⁻¹; CIHRMS (NH₃ gas) calcd for C₁₃H₁₇O₆ (M+H⁺)253.1076, found: 253.1076.

(E)-(1-Propionyl)-4-hydroxy-6-(methyl 6-methyl-hex-2-enoate)-2-pyrone(15 a). To a solution of trimethyl phosphonoacetate (1.7 gm, 9.3 mmol)in THF (20 mL) at room temperature was added NaH (360 mg, 8.95 mmol, 60%dispension in mineral oil). The resulting reaction was stirred at RT for15 min, before aldehyde 5 c (906 mg, 3.58 mmol) in 10 mL THF was addedvia cannula. The reaction was allowed to stir at RT for 3.0 h beforebeing diluted with aqueous NH₄Cl solution (50 mL). The mixture wasextracted with EtOAc (2×100 mL), dried (MgSO₄), filtered, andconcentrated in vacuo. The crude product was flash chromatographed (20%EtOAc in hexanes as eluant) to afford compound 15 a (904 mg, 82% yield)as a yellow oil:

¹H NMR (400 MHz, CDCl₃) δ6.79 (dt,j=6.8, 15.6 Hz, 1H), 5.83 (s, 1H),5.70 (d,j=15.6 Hz, 1H), 3.57, (s, 3H), 2.97 (q, j=7.1 Hz, 2H), 2.49 (m,1H), 2.11 (m, 2H); 1.78 (m, 1H), 1.58 (m, 1H), 1.14 (d,j=6.7 Hz, 3H),1.02 (t,j=7.1 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ208.2, 180.8, 174.8,166.6, 160.8, 147.4, 121.8, 100.0, 99.6, 51.4, 38.2, 35.2, 32.1, 29.5,17.7, 7.6; IR (neat)δ max: 2980, 1726, 1637, 1560, 1438, 1279, 1206,1045, 832 cm⁻¹; CIHRMS (NH₃ gas) calcd for C₁₆H₂₀O₆ (M+H⁺) 308.1260,found: 308.1234.

EXAMPLE 8 Preparation of Compound 14 b/c

(E)-3-Methyl-hex-2-en-1-ol (13 b). To a white slurry of zirconocenedichloride (11.7 g, 40 mmol) in 100 mL of (CH₂)₂Cl₂ was added AlMe₃ (40mL, 2.0M in hexanes, 80 mmol) at 0° C., stirred for 45 min, and thenwarmed to RT for 1.5 h. To this lemon-yellow solution was added1-pentyne 19 a (2.72 g, 40 mmol, dissolved in 20 mL (CH₂)₂Cl₂ at Thereaction was allowed to stir for 3.0 h. The volatile components wereevaporated under reduced pressure (maximum 50° C., 0.3 mm Hg, 2.5 h).The remaining orange-yellow organic residue was extracted with dryhexanes (4×30 mL), and the yellow extract was transferred to a 500 mLround-bottom flask via a cannula. To this was added n-BuLi (16 mL, 2.5 Min hexanes, 40 mmol) at 0° C. This orange-yellow slurry was stirred from0° C. to RT for 1.5 h, and then THF (70 mL) was added to dissolve theprecipitate. The resulting solution (homogeneous, brown-yellow color)was cannulated to a suspension of paraformaldehyde (6.0 g, 200 mmol) inTHF (100 mL) under a N₂ atmosphere. This orange-yellow mixture wasallowed to stir at RT for 20 h before it was cooled to 0° C. (ice waterbath). Ice was added to dilute the reaction, and then saturated NH₄Cl(100 mL) was added. The ice bath was removed and the reaction wasacidified with 3 M HCl until the reaction mixture turned clear yellow(and became homogeneous). At this time, the reaction pH was measured at2˜3. The organic layer was separated, and the aqueous layer wasextracted with ether (2×150 mL). The organic extracts were combined andwashed with a saturated NaHCO₃ solution (200 mL), then dried withNa₂SO₄, filtered and concentrated under reduced pressure to provide thecrude allylic alcohol 13 a. This material was purified by flashchromatography (20% EtOAc in hexanes as eluant) to afford alcohol 13 a(3.37 g, 74% yield) as a colorless oil:

¹H NMR (400 MHz, CDCl₃) δ5.39 (t,j=7.0 Hz, 1H); 4.13 (d,j =7.0 Hz, 2H);1.97 (t,j=7.3 Hz, 2H); 1.64 (s, 3H); 1.4 2(m, 2H), 1.15 (br, 1H), 0.85(t, j=7.3 Hz, 3H); ¹³C NMR (67.5 MHz, CDCl₃) δ139.2, 123.3, 59.0, 59.0,41.5, 20.6, 15.9, 13.6; IR (neat)δ max: 3353, 2959, 1669, 1457,1003cm⁻¹; CIHRMS (NH₃ gas) calcd for C₇H₁₄O₁ (M⁺) 114.1045, found: 114.1035.

(E)-3-Methyl-hept-2-en-1-ol (13 b). To a white slurry solution ofzirconocene dichloride (7.3 g, 25 mmol) in 60 mL (CH₂)₂Cl₂ was addedAlMe₃ (25 mL, 2.0 M in hexanes, 50 mmol) at 0° C., stirred for 30 min,and then warmed to RT for 1.0 h. To this lemon-yellow solution was added1-hexyne 19 b (2.05 gm, 25 mmol, dissolved in 20 mL (CH₂)₂Cl₂ at RT. thereaction was allowed to stir at RT for 16 h. The volatile componentswere evaporated under reduced pressure (maximum 50° C., 0.3 mmHg, 12 h).The remaining orange-yellow organic residue was extracted with dryhexanes (4×30 mL), and the yellow extract was transferred to a 500 mLround-bottom flask via cannula. To this was added n-BuLi (10 mL, 2.5 Min hexanes, 25 mmol) at 0° C. The resulting orange-yellow slurry wasstirred from 0° C. to RT for 1.5 h, and then THF (50 mL) was added todissolve the precipitate. The resulting solution (which was homogeneousand brown-yellow in color) was cannulated to a suspension ofparaformaldehyde (3.75 g, 125 mmol) in THF (50 mL) under a N₂atmosphere. This orange-yellow suspension was allowed to stir at RT for20 h before it was cooled to 0° C. (ice water bath). Ice was added todilute the reaction, and then saturated NH₄Cl (100 mL) was added. Theice bath was removed and the reaction was further acidified with 3 M HCluntil the reaction turned to a clear yellow (homogeneous) solution. Atthis time, the reaction pH was measured as 2-3. The organic layer wasseparated, and the aqueous layer was extracted with ether (2×150 mL).The organic extracts were combined and washed with a saturated solutionNaHCO₃ (200 mL), then dried with Na₂SO₄, filtered and concentrated underreduced pressure to provide crude allylic alcohol 13 b. The crudeproduct was purified by flash chromatography (20% EtOAc in hexanes aseluant) to afford alcohol 13 b. (2.46 g, 77% yield) as a colorless oil:

¹H NMR (400 MHz, CDCl₃) δ5.37 (t,j=7.1 Hz, 1H), 4.11 (d,j=7.1 Hz, 2H),1.98 (t,j=7.5 Hz, 2H), 1.64 (s, 3H), 1.37 (m, 2H), 1.28 (m, 2H), 0,87(t,j=7.3 Hz, 3H); ¹³C NMR (67.5 MHz, CDCl₃) δ139.9, 123.1, 59.2, 39.2,29.8, 22.3, 16.1, 13.9; IR (neat)δ max: 3330, 2958, 2930, 1670, 1467,1000 cm⁻¹; CIHRMS (NH₃ gas) calcd for C₈H₁₆O₁ (M⁺) 128.1201, found:128.1199.

(E)-3-Methyl-hex-2-en-1-al (14 b). To a suspension solution of alcohol13 b (892 mg, 7.82 mmol), 4 Å molecular sieves (4.0 g, activated), andNMO (1.83 g, 15.64 mmol) in CH₂Cl₂ (12 mL) at 0° C., was added TPAP (165mg,0.47 mmol) in one-portion. The resulting dark reaction mixture wasallowed to stir at 0° C. for 30 min, before it was diluted with CH₂Cl₂(20 mL), and then filtrated through a short pad of silica gel. Thecluent was concentrated in vacuo to afford aldehyde 14 b (823 mg, 94%yield) as a viscous, colorless oil. This unstable aldehyde wassufficiently pure, and used immediately without further purification:

¹H NMR (400 MHz, CDCl₃) δ9.98 (d,j=8.2 Hz, 1H), 5.85 (d,j=8.2 Hz, 1H),2.17 (t,j=7.3 Hz, 2H), 2,14 (s, 3H), 1.52 (m, 2H), 0.91 (t,j=7.3 Hz,3H); ¹³C NMR (67.5 MHz, CDCl₃) δ191.7, 164.5, 127.8, 43.0, 20.7, 17.8,14.0; IR (neat)δ max: 2961, 2932, 2870,1678, 1458,1190,1131 cm⁻¹.

(E)-3-Methyl-hept-2-en-1-al (14 c). To a suspension solution of alcohol13 b (739 mg, 5.7 mmol), 4 Å molecular sieves (2.9 g, activated), andNMO (1.33 g, 11.4 mmol) in CH₂Cl₂ (12 mL) at 0° C., was added TPAP (120mg,0.34 mmol) in one-portion. The resulting dark reaction mixture wasallowed to stir at 0° C. for 30 min, before being diluted with CH₂Cl₂(20 mL), and then filtered through a short pad of silica gel. Thefiltrate was concentrated in vacuo to afford aldehyde 14 c (704 mg, 98%yield) as a colorless oil. The aldehyde is sufficiently pure forimmediate use without further purification:

¹H NMR (400 MHz, CDCl₃) δ9.94 (d,j=8.1 Hz, 1H), 5.82 (d,j=8.1 Hz, 1H),2.16 (t,j =7.6 Hz, 2H), 2.11 (s, 3H), 1.44 (m, 2H), 1.28 (m, 2H), 0.87(t,j=7.3 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ191.3, 164.5, 127.2, 40.3,29.2, 22.3, 17.4, 13.8; IR (neat)δ max: 2959, 2933, 2863, 1676, 1467,1195, 1131 cm⁻¹.

EXAMPLE 8 Preparation of Myxopyronin A/B

3-[(E,E)-2,5-Dimethyl-2,4-octadienoyl]-4-hydroxy-6-(methyl 6methyl-hex-2-enoate)2-pyron (17 c). Compound 15 a (58 mg, 0.188 mmol)was dissolved in CH₂Cl₂ (2.5 mL) and stirred at −78° C. under argonatmosphere. To this yellow solution was added freshly distilled TiCl₄(82 μl, 0.75 mmol), the reaction turned to an orange-yellow slurrymixture immediately. After stirring for 45 min at −78° C., DIPEA (144μL, 0.83 mmol) was added, and the reaction became a red-dark reactionmixture. This reaction mixture was allowed to stir at −78° C. for 4.0 h,then aldehyde 14b (84 mg, 0.75 mmol) dissolved in 1.0 mL CH₂Cl₂ wasadded via a cannula. This dark red reaction mixture was stirred at −78°C. for 50 h and then 0° C. for 5-10 min, before it was quenched withdistilled water. The reaction was extracted with CH₂Cl₂ (3×20 mL), dried(Na₂SO₄), and concentrated in vacuo. The crude product was flashchromatographed (20% EtOAc in hexanes as eluant) to provide diene 17 c(44 mg, 58% yield) as a sticky yellow oil:

¹H NMR (400 MHz, CDCl₃): δ6.97 (d,j=11.4 Hz,1H), 6,89 (dt,j=6.5, 15.3Hz, 1H), 6.13 (d,j=11.4 Hz, 1H), 5.92 (s, 1H); 5.83 (d,j=15.3 Hz, 1H),3.70 (s, 3H), 2.58 (m, 1H), 2.21 (m, 2H), 2.13 (t,j=7.3 Hz, 2H), 1.98(s, 3H), 1.90 (m, 1H), 1.82 (s, 3H), 1.66 (m, 1H), 1.48 (m, 2H), 1.24(d,j=7.3 Hz, 3H), 0.89 (t,j=7.3 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃):δ201.6, 180.7, 174.7, 166.7, 160.3, 149.1, 147.5, 133.6, 132.9, 121.9,120.6, 100.0, 99.1, 51.5, 42.8, 38.2, 32.2, 29.6, 21.0, 17.7, 17.2,13.8, 13.5; IR (neat)δ max: 2958, 1726, 1637, 1547, 1436, 1383, 1329,1266, 1205, 1044, cm⁻¹; CIHRMS (NH3 gas) calcd for C₂₃H₃₁O₆ (M=H⁺)403.2120, found: 403.2108.

3-[(E,E)-2,5-Dimethyl-2,4-nonadienoyl]-4-hydroxy-6-(methyl6-methyl-hex-2-enoate)2-pyron (17 d). Compound 15 a (178 mg, 0.578 mmol)was dissolved in CH₂Cl₂ (10 mL) and stirred at −78° C. under argonatmosphere. To this was added freshly distilled TiCl₄ (254 μl, 2.31mmol); the reaction became a yellow slurry immediately. After stirringfor 20 min at −78° C., DIPEA (483 μL, 2.77 mmol) was added, and thereaction mixture became dark red. After stirring at −78° C. for 3.0 h,aldehyde 14 c (254 mg, 2.0 mmol) dissolved in 2.0 mL CH₂Cl₂ was addedvia cannula. The dark red reaction mixture was stirred at −78° C. for 48h and then warmed to 0° C. for 5 min, before being quenched withdistilled water. The reaction mixture was extracted with CH₂Cl₂ (3×20mL). The organic layer was dried (Na₂SO₄), filtered, and concentrated invacuo. The crude product was flash chromatographed (25% EtOAc in hexanesas eluant) to provide diene 17 d (147 mg, 61% yield) as a sticky yellowoil:

¹H NMR (400 MHz, CDCl₃): δ6.97 (d,j =11.6 Hz, 1H), 6.88 (dt,j=6.7, 15.3Hz, 1H), 6.13 (d,j=11.6 Hz, 1H), 5.91, (s 1H), 5.81 (d,j=15.3 Hz, 1H),3.70 (s, 3H), 2.58 (m, 1H), 2.20 (m, 2H), 2.14 (t,j=7.3 Hz, 2H), 1.98(s, 3H), 1.88 (m, 1H), 1.82 (s, 3H), 1.66 (m, 1H), 1.45 (m, 2H), 1.30(m, 2H), 1.24 (d,j=6.7 Hz, 3 H), 0.89 (t,j=7.3 Hz, 3H); ¹³C NMR (75 MHz,CDCl₃) δ201.6, 180.7, 174.7, 166.7, 160.3, 149.4, 147.5, 133.6, 132.9,121.9, 120.4, 100.0, 99.1, 51.5, 40.5, 38.2, 32.2., 30.0, 29.6, 22.4,17.7, 17.3, 14.0, 13.5; IR (neat)δ max: 2932, 2872, 1726, 1636, 1546,1436, 1383, 1329, 1268, 1206, 1175, 1045 cm⁻¹; CIHRMS (NH₃ gas) calcdfor C₂₄H₃₂O₆ (M⁺) 416.2199, found: 416.2208.

3[(E,E)-2,5-Dimethyl-2,4-octadienoyl]-4-hydroxy-6-(6-methyl-hex-2-enoicacid)-2-pyrone (21 a). To a stirred solution of 17 c (60 mg, 0.15 mmol)in THF (8.0 mL) was added LiOH aqueous solution (2.0 mL, 1.0 M, aq., 2.0mmol) at RT, the resulting reaction mixture (THF/H₂O=4:1) was allowed tostir at RT for 20 h before it was diluted with EtOAc (10 mL), and thenquenched by saturated NH4Cl solution (10 mL). The mixture was acidifiedto pH 2 by slow addition of 5% HCl. The solution was extracted withEtOAc (3×15 mL), dried (Na₂SO₄), filtered through a short pad of silicagel, and concentrated in vacuo to afford the crude acid 21 a (58 mg,100% yield) as a yellow, sticky oil. This material was used in thesubsequent reaction without further purification:

¹H NMR (400 MHz, CDCl₃): δ7.03-6.96 (m, 2H), 6.14 (d,j=11.6 Hz, 1H),5.92 (s, 1H), 5.82 (d,j=15.9 Hz, 1H), 2.59 (m, 1H), 2.24 (m, 2H), 2.13(t,j=7.6 Hz, 2H), 1.98 (s, 3H), 1.89 (m, 1H), 1.82 (s, 3H), 1.68 (m,1H), 1.48 (m, 2H), 1.30 (m, 2H), 1.24 (3,j=6.7 Hz, 3H), 0.89 (t,j=7.3Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ201.6, 180.7, 174.5, 171.4, 160.4,150.2, 149.1, 133.6, 132.9, 128.3, 121.6, 120.6, 100.1, 9.1, 42.8, 38.2,32.0, 29.7, 20.9, 17.7, 17.2, 13.8, 13.4; IR (neat)δ max: 2960, 1724,1636, 1561, 1446, 1383, 1249, 973, 831 cm⁻¹; CIHRMS (NH₃ gas) calcd forC₂₂H₂₉O₆ (M+H⁺) 389.1964, found: 389.1968.

3-[E,E-2,5-Dimethyl-2,4-nonadienoyl]-4-hydroxy-6-(6-methyl-hex-2-enoicacid)-2-pyrone (21 b). To a stirred solution of 17 d (42 mg, 9.10 mmol)in THF (6.0 mL) was added LiOH aqueous solution (1.5 mL, 1.0M aq., 1.5mmol) at RT, the resulting reaction mixture (THF/H₂O=4:1) was allowed tostir at RT for 35 h before it was diluted with EtOAc (10 mL) and thenquenched with saturated NH₄Cl aq. (10 mL). The reaction mixture wasacidified to pH 2 by slow addition of 5% HCl, and extracted with EtOAc(3×15 mL). The organic layer was dried (Na₂SO₄), filtered through ashort pad of silica gel, and then concentrated in vacuo to afford thecrude acid 21 b (40 mg, 100% yield) as a sticky yellow oil. Thismaterial was used without further purification:

¹H NMR (270 MHz, CDCl₃): δ7.05-6.94 (m, 2H), 6.14 (d,j=11.5 Hz, 1H),5.92 (s, 1H), 5.81 (d,j=15.4 Hz, 1H), 2.59 (m, 1H), 2.24 (m, 2H), 2.14(t,j=7.3 Hz, 2H), 1.97 (s, 3H), 1.89 (m, 1H), 1.82 (s, 3H), 1.68 (m,1H), 1.43 (m, 2H), 1.28 (m, 2H), 1.24 (d,j=6.8 Hz, 3 H), 0.89 (t,j=7.3Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ201.6, 180.7, 174.5, 171.4, 160.4,150.2, 149.1, 133.6, 132.9, 128.3, 121.6, 120.6, 100.1, 99.1, 42.8,38.2, 32.0, 29.7, 20.9, 17.7, 17.2, 13.8, 13.4; IR (neat)δ max: 3434,2960, 1724, 1636, 1561, 1446, 1383, 1249, 973, 831; IR (neat)δ max:2931, 1697, 1637, 1544, 1439, 1383, 1249, 972, 914 831 cm⁻¹; CIHRMS (NH₃gas) calcd for C₂₃H₁₃O₆ (M+H⁺) 403.2121, found: 403.2136.

(±)-Myxopyronin A (18). To a stirred solution of acid 21 a (34 mg,0.0876 mmol) in dry acetone (1.5 mL) was added DIPEA (37 μL, 0.21 mmol)and then ethyl chloroformate (18 μL, 0.193 mmol) at 0° C. The reactionmixture was stirred at 0° C. for 1.5 h, then NaN₃ (17 mg, 0.263 mmol,dissolved in 300 μL distilled H₂O) was added via syringe. The resultingreaction mixture was stirred at 0° C. for 45 min before being dilutedwith ice water (5 mL). The reaction mixture was extracted with distilledtoluene (8×5 mL), dried, (MgSO₄), filtered, and concentrated in vacuo.The organic residue was taken up by dry toluene (6 mL) and refluxed for2.5 h before fresh distilled MeOH (3.0 mL) was added to trap theisocyanate intermediate. The resulting solution was refluxed for anadditional 8.0 h and then concentrated in vacuo to provide crude(±)-myxopyronin A (18) as a yellow oil. The crude material was purifiedby preparative reversed phase HPLC (70:30:4 MeOH/H₂O/AcOH) to providepure (±)-myxopyronin A (18, 26 mg, 71%) as a sticky yellow oil:

¹H NMR (400 MHz, CD₃OD, 3.31 ppm) δ7.17 (d,j=11.6 Hz, 1H), 6.40(d,j=14.0 Hz, 1H), 6.27 (d,j=11.6 Hz, 1H), 6.08 (s,1H), 5.04 (dt,j=7.3,14.0 Hz, 1H), 3.66 (s, 3H), 2.66 (m, 1H), 2.20 (t,j=7.3 Hz, 2H), 2.01(m, 2H), 1.94 (s, 3H), 1.81 (s, 3H), 1.76 (m, 1H), 1.59 (m, 1H), 1.53(m, 2H), 1.25 (d,j=6.7 Hz, 3H), 0.93 (t,j=7.6 Hz, 3H); ¹³C NMR (75 MHz,CD₃OD, 49.15 ppm) δ199.0, 174.9, 173.5, 164.8, 156.9, 151.4, 138.3,135.2, 126.2, 122.4, 110.7, 102.5, 101.4, 52.8, 44.0, 44.0. 39.3, 35.9,28.7, 22.2, 18.6, 17.4, 14.2, 12.0; IR (neat)δ max: 3313, 2931, 1717,1681, 1636, 1537, 1439, 1381, 1247, 1052, 953 cm⁻¹; UV (methanol): max(log ξ)=213, 298 nm; CIHRMS (NH₃ gas) calcd for C₂₃H₃₂N₁O₆ (M=H⁺)418.2230, found: 418.2198.

(±)-Myxopyronin B (18 a). To a stirred solution of acid 21 b (22 mg,0.0547 mmol) in dry acetone (1.0 mL) was sequentially added DIPEA (23μL, 0.13 mmol) and ethyl chloroformate (11 μL, 0.12 mmol) at 0° C. Thereaction mixture was stirred at 0° C. for 1.5 h, then NaN₃ (17 mg, 0.263mmol, dissolved in 300 μL distilled H₂O) was added via syringe. Theresulting mixture was stirred at 0° C. for 70 min before being quenchedwith ice water (3 mL), and extracted with distilled toluene (6×5 mL).The organic layer was dried (MgSO₄), filtered, and concentrated invacuo. The organic residue was taken up with dry toluene (6 mL), andrefluxed for 2.0 h before freshly distilled MeOH (3.0 mL) was added totrap the isocyanate intermediate. The resulting solution was furtherrefluxed for 12 h and then concentrated in vacuo to provide crude(±)-myxopyronin B (18 a) as a yellow oil. This crude material waspurified by preparative reversed phase HPLC (70:30:4 MeOH/H₂O/AcOH) toprovide pure (±)-myxopyronin B (18 a, 15.6 mg, 66%) as a yellow stickyoil:

¹H NMR (400 MHz, CD₃OD, 3.27 ppm) δ7.15 (d,j=11.6 Hz, 1H), 6.36(d,j=14.0 Hz, 1H), 6.23 (d,j=11.6 Hz, 1H), 6.01 (s, 1H), 5.02 (dt,j=7.3,14.0 Hz, 1H), 3.62 (s, 3H), 2.61 (m, 1H), 2.18 (t,j=7.3 Hz, 2H), 1.97(m, 2H), 1.90 (s, 3H), 1.78 (s, 3H), 1.72 (m, 1H), 1.55 (m, 1H), 1.49(m, 2H), 1.31 (m, 2H), 1,21 (d,j=7.3 Hz, 3H), 0.90 (t,j=7.0 Hz. 3H); ¹³CNMR (75 MHz, CD₃OD, 49.15 ppm) δ199.1, 175.2, 173.2, 165.0, 156.9,151.6, 138.4, 135.2, 126.1, 122.3, 110.7, 102.6, 101.9, 52.8, 41.6,39.2, 35.9, 31.3, 29.7, 23.6, 18.6, 17.4, 14.4, 11.9; IR (neat) max:3315, 2931, 1734, 1681, 1635, 1539, 1448, 1382, 1237, 1051, 953 cm⁻¹; UV(methanol): λmax (log ξ)=213, 298 nm; CIHRMS (NH₃ gas) calcd forC₂₄H₃₄N₁O₆ (M+H⁺) 432.2386, found: 432.2377.

EXAMPLE 9

In vitro transcription reactions. [α-³²P] UTP-incorporated RNA wassynthesized in 50 μl reaction volumes containing transcription buffer(50 mM Tris-HCl, pH 8.0, 200 mM KCl, 10 mM MgCl2, 10 mM DTT and 1.5 μMBSA), 1 μg of DNA template, 4 μM UTP containing 5 μCi of [α-³²P] UTP,400 μM each of ATP, GTP, and CTP. After incubation for 60 minutes at 25°C., the reaction is terminated with 100 μl 100% TCA, which alsoprecipitates the newly transcribed RNA.

Microdilution Minimal Inhibitory Concentration (MIC) and MinimumBactericidal Concentration (MBC) Assays

The minimal inhibitory concentration (MIC) is defined as the lowestconcentration of antimicrobial agent that completely inhibits growth ofthe organism in the microliter plate. The MIC is reported as a rangebetween the concentration at which no growth is observed and theconcentration of the dilution which immediately followed. Selectedinhibitors from the RNA polymerase screen described above were testedfor their ability to inhibit bacterial growth in a broth microdilutionassay as follows. Mueller-Hinton broth containing 20-25 mg/L Ca2+ and10-12.5 mg/L Mg2+ (Difco #0757-07-8) is recommended as the medium (pH7.2 and 7.4 at room temperature) of choice by the NCCLS for rapidlygrowing or facultative organisms and it demonstrates good batch-to-batchreproducibility for susceptibility testing; is low in sulfonamide,trimethoprim, and tetracycline inhibitors; and yields satisfactorygrowth of most pathogens. Dilution of antimicrobial agents is performedin a sterile, covered 96-well microliter plate with flat bottom wells(Costar #9017), and each well contains 100 μL of both +/− antimicrobialagent. The final concentrations of the small molecule antimicrobialagents are 100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.20, 0.10,and 0.05 μg/mL, respectively. Different dilutions are performed fornatural product extracts. They are first diluted 100-fold withMueller-Hinton broth. The final dilutions of the natural productextracts are 200, 400, 800, 1600, 3200, 6400, 12800, 25600, 51200, 1×105, 2×105, and 4×105-fold. A 1% DMSO (no-drug) row is prepared inMueller-Hinton broth as a control for 100% growth on each plate. AMueller-Hinton broth only with no bacteria growth is also included as anegative control for each plate. Ampicillin and rifampin are used aspositive controls against all bacterial strains in every experiment.

The overnight culture of a single colony is diluted in sterileMueller-Hinton broth so that, after inoculation, each well containsapproximately 5×105 CFU/mL. Within 15 minutes of preparation, 50 mL ofthe adjusted inoculum suspension is added to the microliter plate. Eachwell is diluted with an equal volume of the antimicrobial agent/controlsubstance diluted with sterile Mueller-Hinton broth. The inoculatedmicroliter plate is incubated at 35° C. for 16-20 hours. The turbidityof each well is determined by measuring the absorbance at 595 nm on theBioRad Model 3550-UV microplate reader. The rows containing broth only(no cells) serve as a control, and the rows containing the titration of1% DMSO serve as a control for 100% growth. The average of the brothonly controls is subtracted from the average of each duplicate. Thisvalue is subsequently normalized to the average of the DMSO controls.

The minimum bactericidal concentration (MBC) is defined as theconcentration of antimicrobial agent from which no colonies grow onpetri plates or in the medium. In practice, the MBC is arbitrarilydefined as the concentration at which a 1000-fold reduction in colonyforming units is observed with respect to the original inoculum(survival of 0.1%). The broth dilution method consists of inoculatingthe wells from an MIC microliter plate using a 96-well inoculation gridinto a fresh microliter plate containing 100 μL Mueller-Hinton broth perwell. The MBC plates are incubated at 37° C. for 16-20 hrs and the MBCvalues are determined.

The MIC data suggest that these compounds, like rifampicin, do notpenetrate E. coli efficiently since it does inhibit the growth of apermeabilized E. coli. An attractive feature of this series is theactivity against strains that are resistant to rifampicin. The MIC forrifampicin is 10 μM against susceptible strains, but inhibitory activityis greatly reduced against rifampicin resistant strains (>100 μM),illustrating the limitation of rifampicin and the need for discovery ofnew agents. Myxopyronins, however, are equiactive against rifampicinsusceptible and rifampicin resistant S. aureus (FIGS. 20(a) and (b).

Discussion

The total synthesis of myxopyronin A was approached in several ways. Thefirst is shown in the retrosynthesis provided in FIG. 2.

Attachment of the 3-position side chain was accomplished through analdol condensation. Ester 1 is available from 2-pentanone throughWadsworth-Emmons chemistry (Wadsworth, W., Emmons, W., Org. Synth.,1965, 45, 44) and commercially available triethylphosphonates (FIG. 3).The anion of triethyl phosphonacetate is created by stirring with NaH inTHF, and is condensed with 2-pentanone to create unsaturated ester 16.The ester 16 is reduced with DIBAL to the alcohol and then oxidized tothe aldehyde 14 with DDQ. Compound 1 is produced by going throughanother cycle of Wadsworth-Emmons chemistry, DIBAL reduction and DDQoxidation. Intermediate 13 can also be made using lithium dimethylcuprate chemistry (Sum and Weiler, Can. J. Chem., 1979, 57, 1431), whichprocedure produces 13 in a higher E/Z ratio.

The other half of the molecule containing the 6-substituted pyrone isdissected in the following manner. This scheme requires a pyronefunctionalized at the 6-position (Douglas, J., Money, T., Can. J. Chem.,1968, 46, 695) and commercially available reagents shown in FIG. 2. Thethree necessary segments can be joined by an additional Wadsworth-Emmonsolefination and a simple alkylation. The terminal methyl carbamate canthen be introduced by the use of a modified Curtius rearrangement.Overman, L., Taylor, G., Petty, C., Jessup, P., J. Org. Chem., 1978, 43,2164.

Two different pathways are provided for the synthesis of compound 10.The first pathway is based on the known alkylation of the commerciallyavailable compound 3 at the 7-postion using n-BuLi and an alkyl halide.Groutas, W., Stanga, M., Brubaker, M., Huang, T., Moi, M., Carroll, R.,J. Med. Chem., 1985, 28, 1106. (see FIG. 4) A model pyrone containing anethyl group was alkylated at the 7-position in a similar manner byconstructing the 6-position side chain after the desired methyl group(C-8) was already in place at C-7. (The synthetic approach used tosynthesize the 6-ethyl pyrone was developed by Cook and co-workers.Cook, L., Ternai, B., Ghosh, P., J. Med. Chem., 1987, 30, 1017.) Thepresent invention provides a means of installing alkyl chains of varyingsizes at the 6-position by dimerization of various ethyl malonates andsubsequent deacylation of the 3-position (FIGS. 5 and 15). Thereby,pyrone 6 is prepared where R is ethyl.

Pyrone 6 is then alkylated with 3-bromopropionaldehyde dimethyl acetaland the 4-hydroxyl group protected as its SEM ether (FIGS. 6a and 6 b).The dimethyl acetal is removed with dilute H₂SO₄. The Wadsworth-Emmonsreaction (Wadsworth, W., Emmons, W., Org. Synth., 1965, 45, 44)introduces the unsaturated ester, and the SEM group is removed with TBAFto produce key intermediate 10. The 3-position side chain is introducedby use of an aldol reaction. The ester on the 6-position side chain ishydrolyzed, and a Curtius rearrangement installs a vinyl carbamatemoiety to complete the synthesis of myxopyronin A.

In a preferred embodiment, the present invention provides an improvedroute to intermediate 10. Allyl bromide alkylates pyrone 6 in a higheryield (FIG. 7). This modification combined with the use of dilute H₂SO₄in place of TBAF to remove the SEM group substantially increases theoverall yield of 10.

FIGS. 8a and 8 b demonstrate schematically the completion of themyxopyronin A synthesis from key intermediate 10. Aldol condensationwith 1 to produce 13 is followed by oxidation with any reagent effectivefor oxidizing alcohols at allylic or benzylic positions, including DDQ,MnO₂, K₂CrO₇, etc., to afford ketone 17.

Conversion of the ethyl ester to the acid is effected by saponificationwith LiOH. Finally, installation of the vinyl carbamate is effected bymodified Curtius conditions.

In addition, the present invention provides an alternate pathway for thetotal synthesis of myxopyronin A. Formation of the bond between C-3 andC-15 has proven to be a difficult synthetic step. A study of alternativeways of appending the 3-position side chain demonstrated that attachmentof a portion of the 3-position alkyl chain is effectively performed byacylation of the pyrone. This process is based on acylation of6-alkyl-4-hydroxy-2-pyrones by acyl chlorides. Cook, L., Ternai, B.,Ghosh, P., J. Med. Chem., 1987, 30, 1017. However, acylation of pyrone10 with acyl chloride 19 using conditions set forth by Cook (FIG. 9) didnot provide the expected product: no evidence of the double bonds wereobserved by NMR; only compound 10 was recovered.

Acylation of pyrones is successful when acid chlorides with saturatedalkyl chains are used. A retrosynthetic analysis of the preparation ofmyxopyronin A taking advantage of this reaction is shown, in FIGS. 10aand 10 b . Pyrone 10 was synthesized as shown in FIGS. 5-7. At thispoint, the pyrone is acylated with propionyl chloride. The rest of the6-position side chain is then attached using a base-catalyzed aldolreaction with aldehyde 14, an intermediate in the synthesis of 1 (FIG.3) and the 3-propionyl pyrone 15. The synthesis is completed with aCurtius rearrangement as illustrated in FIGS. 8a and 8 b.

The alternate route works effectively as disclosed herein. Pyroneintermediate 10 is acylated with propionyl chloride in TFA to givecompound 15. An aldol reaction using LDA in THF condenses aldehyde 14with 15. Subsequent treatment with MsCl and DBU gives the desired dienein the side chain and yields intermediate 17.

Analogs of Myxopyronins

As will be apparent to one of skill in the art, various isomers(including stereoisomers), analogs, and derivatives of the pyronins aremade accessible by the subject invention by appropriate modification.For example, the total synthesis of myxopyronin B is easily carried outby simple modification of the pathway shown in FIG. 3, i.e., bysubstituting 2-hexanone for 2-pentanone at the beginning of thesynthesis. As disclosed herein, the synthetic pathways provide a mixtureof enantiomers which can be prepared in purified form by any of avariety of methods well known in the art, including but not limited tochiral HPLC, resolution of one of the intermediates set forth herein, orseparation of a diastereomeric derivative. In addition, the routes maybe modified by those of ordinary skill in the art to provide derivativesfrom the isocyanate intermediate other than carbamates. For example,instead of reacting with an alcohol, reaction with ammonia or analkylamine would lead to a substituted urea analog. Satchell, Chem. Soc.Rev., 1975, 4, 231. Also, hydrolysis generates a primary aminemyxopyronin analog. cl Convergent Synthesis of Myxopyronin A

Pyrones have been used to elicit a biological effect in a few instancesbut in none of them have they been used as an antibacterial agent.2H-Pyran-2,6(3H)-dione derivatives are reported to be active atreasonable doses in a passive cutaneous anaphylaxis model in rats whenadministered by either the intravenous or oral route. Snader, K. M. etal., J. Med. Chem., 1979, 22, 706; Chahrin, L. W., Snader, K. M.,Williams, C. R., 2H-Pyran-2,6(3H)-dionederivate, German Patent 25 33843. In a second case, simple 3-(1-oxoalkyl)-4-hydroxy-6-alkyl-2-pyroneswere found to be effective in vitro in the inhibition of human sputumelastase. Cook, L., Ternai, B., Ghosh, P., J. Med. Chem., 1987, 30,1017. Lastly, a series of coumarin derivatives were found to beeffective inhibitors of HIV protease in both enzymatic assays and cellculture (FIG. 1(b)). Skulnick, H. I., et al., J. Med. Chem., 1995, 38,4968. No synthetic investigations of pyronin antibacterials have beenreported in the literature. The total synthesis of myxopyronin A wasapproached in a highly convergent manner. The retrosynthesis ofmyxopyronin A is shown in FIGS. 11a and 11 b.

Preparation of compound 14 (FIG. 12(a)) was started from commerciallyavailable 1-pentyne. Regioselective and ster-eospecific carboaluminationof 1-pentyne in the presence of 2.0 equiv of AlMe₃and 1.0 equiv ofCp₂ZrCl₂ (Cp=η—C₅H₅) afforded the organoalane species, which was treatedwith 1.0 equiv of ^(n-)BuLi, and quenched with paraformaldehyde to givegeometrically pure (E) allylic alcohol 13 a with 74% yield. Swernoxidation of alcohol 13 a cleanly generated versatile aldehyde 14, whichis not very stable and used immediately after flash chromatography.

A synthetic approach to pyrone 5 (R=Et) (FIG. 12(b)) was based on aknown procedure reported by Cook and co-workers (supra). Commerciallyavailable ethyl propionyl-acetate was hydrolyzed under basic conditions(1.5 M aqueous NaOH) to provide acid 4 a (76% yield), which wasdimerized, in the presence of carbonyidiimidazole, to afford pyrone 5(R=Et) in 98% yield. This sequence of reactions easily provided aquantitative amount of pyrone 5 (R=Et), which is the core structure inthe myxopyronin A structure.

Preparation of compound 8 b (FIG. 12(c)) is straight forward.Monoprotection of 1,3-propane diol was achieved by using 1.0 equiv ofNaH and 1.0 equiv of TBSCl, and it cleanly generated alcohol 8 a in 98%isolated yield. lodination of alcohol 8 a under the condition ofI₂/PPh₃/imidazole provided iodide 8 b as a single product with 96%yield.

Subsequent research has led to an improved synthetic approach to theintermediate 15 (FIG. 13(a)). Pyrone 5 (R=Et) was lithiated (3.2 equivof LDA) at −78° C., the derived anion was treated with iodide 8 b toafford compound 5 a as a single alkylated product in 80-87% isolatedyield. Compound Sa was deprotonated (AcOH/THF/H₂O, 2:2:1 mixturesolution) at RT to provide alcohol 5 b (91% yield), which was oxidizedin the presence of the Dess-Martin periodinate reagent to yield aldehyde5 c. Wadsworth-Emmons homologation of aldehyde 5 c (triethylphosphonoacetate, NaH, C₆H₆) cleanly generated α,β-unsaturated ester 15as a single isomer with a 89% yield. FIG. 13(b) schematicallydemonstrates the completion of the myxopyronin A synthesis from keyintermediate 15.

Aldol condensation of ethyl ketone 15 with aldehyde 14 in the presenceof TiCl₄ was investigated. Titanium enolate was generated at −78° C. bythe treatment of ethyl ketone 15 with 4.0 equiv of TiCl₄ and 4.4 equivof Et₃N. The derived (Z)enolate was condensed with an excess of freshlyprepared aldehyde 14 to produce diol 14 a in 71.3% isolated yield.Treatment of diol 14 a with methane sulfonyl chloride, followed byelimination mediated by DBU, furnished the trisubstituted diene 17 in a72% yield. Conversion of 17 to the α,β-unsaturated acid 17 a wasaccomplished with LiOH. Finally, installation of the vinyl carbamatefunctional group was effected through modified Curtius condition. Acid17 a was combined with diphenylphosphoryl azide (PhO₂P(O)N₃) and Et₃Nand refluxed in C₆H₆ for 9.0 h. The derived azide intermediate wastreated with anhydrous MeOH and was refluxed for another 6.0 h toproduce (±)myxopyronin A, compound 18.

Myxopyronin A Synthesis Aldolization of an Acyl Pyrone

Acylation of pyrones at the 3-position is successful when the acidchlorides of short, saturated alkyl chains are used. Douglas, J., Money,T., Can. J. Chem., 1968, 46, 695. Acylation of 10 with propionylchloride is followed by a base-catalyzed aldol with the appropriatealdehyde to attach the rest of the native side-chain. The necessaryaldehyde 14 (FIG. 14) is generated by way of the ester 16. (Sum, F. W.,Weiler, L., Can. J. Chem., 1979, 57, 431) Reaction of ethylbutyrylacetate with diethyl chlorophosphite and LiMe₂Cu gavepredominantly the E, isomer of 16 after distillation. DIBAL reduction tothe alcohol followed by Swern oxidation produced the aldehyde 14.

With aldehyde 14 in hand, the route shown in FIGS. 11a and 11 b was usedto complete the synthesis of myxopyronin A. Pyrone intermediate 10 isacylated with propionyl chloride in TFA to give compound 15. The overallconversion is efficient since unreacted starting material can berecovered by chromatography. An aldol reaction between the pureregioisomer of 14 and 15 was performed using LDA in THF. The crudealcohol was converted to the mesylate and then eliminated using DBU.This yielded the desired diene 17.

To complete the synthesis, 17 was saponified to the acid using LiOH. Amodified Curtius sequence (Overman, L., Taylor, G., Petty, C., Jessup,P., J. Org. Chem., 1978, 43, 2164) was then used to install the unusualvinyl carbamate moiety and thereby afford the desired myxopyronin A. Theidentity of the product was confirmed by comparison of spectral data forthe synthetic product with that of an authentic sample of the naturalproduct. Resolution of the enantiomers generated at C-7 may be achievedby means of chiral HPLC.

Synthesis of Myxopyronin A and B

The absolute stereochemistry of myxopyronin A and B (Kohl, W., et al.,Liebigs Ann. Chem. 1983, 1656-1667; Kohl, W., et al., Liebigs Ann. Chem.1984, 1088-1093; Jansen, R., et al., Liebigs Ann. Chem. 1985, 822-836)has been determined by careful degradative and spectroscopic methods andwas assigned as the (R)-configuration¹ (FIG. 16). Riechenbach determinedthose molecules to be broad spectrum antibiotics and selectiveinhibitors of bacterial DNA-dependent RNA polymerase. lrschik, H., etal., J. Antibiot. 1983, 36, 1651-1658. The broad spectrum of activityand selectivity for bacterial RNA polymerase over the human polymeraseestablished the myxopyronins as promising candidates for development asantibacterial agents. The exhibition of activity against rifampicin orstreptolydigin resistant bacteria by the myxopyronins suggest that theseagents target a region of RNA polymerase distinct from the one byrifampicin. No prior synthetic methods have been reported concerningthis class of natural products. The present convergent synthesis makesuse of a 3-propionyl-4-hydroxy-α-pyrone as the central building blockfrom which both side chains are introduced. An alkylation strategy wasused for the installation of the lower side chain followed by atitanium(IV) promoted aldol condensation introducing the (E,E)-dienoneof the upper side chain. A retrosynthetic analysis of the myxopyroninsis illustrated in FIG. 16 with the first disconnection removing theterminal unsaturated carbamate. This detachment produced the advancedintermediate 21 bearing an unsaturated carboxylate functionality. Thesecond disconnection produced the aldol synthons in the form of thepyrone 15 a and the unsaturated aldehydes. Further disconnection of 15 aproduced the starting 3-propionyl-4-hydroxy-α-pyrone 5. (Pyrone 5 wasprepared from commercial available ethyl propionate see: Cook, L., etal., J. Med. Chem. 1987, 30, 1017-1022.)

Preparation of the O1-C12 Fragment. The synthesis of this materialrelied on a selective alkylation of the C6 ethyl group of pyrone 5 (FIG.17). In the presence of LDA (3.2 equiv), the regioselective alkylationwith primary iodide 8 c proceeds through the trianion intermediate togive the alkylated pyrone 5 a in good yield. Intermediate 5 a wasdeprotected under mild acidic conditions to afford the correspondingprimary alcohol in 90% yield. (Alkyl iodide 8 c was prepared from1,3-propane diol by a two step reaction sequence: (i) selectiveprotection with TBSCl (1.0 equiv), NaH (1.0 equiv), 98% yield; (ii) I₂,PPh₃, imidazole, 96% yield; satisfactory spectroscopic data (¹H and¹³C-NMR, IR, CIMS and CIHRMS) were obtained for all new compounds.)Oxidation of the primary hydroxyl with freshly prepared Dess-Martinreagent (2.0 equiv, 0° C., 2 h) gave aldehyde 5 c. (Dess, D. B.; Martin,J. C. J. Org. Chem. 1983, 48, 4155-4156; Dess, D. B.; Martin, J. C. J.Am. Chem. Soc. 1991, 113, 7277-7287. Improved procedures for thepreparation of DMP are found in Ireland, R. E.; Liu, L. J. Org. Chem.1993, 58, 2899, and Meyer, S. D.; Schreiber, S. L. J. Org. Chem.1994,59, 7549-7552.) Subsequent Horner-Emmons-Wadsworth homologationusing trimethyl phosphonoacetate (2.2 equiv NaH, 2.2 equiv THF, rt)afforded the α,β-unsaturated ester 15 a with a E:Z isomer ratio greaterthan 20:1, after silica gel chromatography to provide geometrically pure15 a in 82% yield. This sequence completed the preparation of the O1-C12fragment now set for the subsequent aldol condensation for theintroduction of the C15-C24 and C25 side chains of myxopyronin A and B.

Synthesis of the α,α-Unsaturated Aldehydes 14 b and 14 c. The synthesisof these subunits relied on the Negishi's carbon alumination of theterminal alkynes 19 a and 19 b (FIG. 18). Negishi, E., et al., J. Am.Chem. Soc. 1996, 118, 9577-9588; Okukado, N.; Negishi, E. TetrahedronLett. 1978, 27, 2357-2360. Treatment of alkyne 19a/b with zirconocenedichloride (1.0 equiv) in the presence of AlMe₃ (2.0 equiv) afforded the(E)-trisubstituted vinyl aluminate 20, which was then directly convertedto the more reactive aluminate complex by addition of n-BuLi (1.0 equiv,THF). This intermediate was trapped with excess paraformaldehydeaffording the (E)-trisubstituted allylic alcohol 13 b/c in good overallyield. Oxidation of this material with TPAP (0.06 equiv;tetrapropylammonium perruthenate; Ley, S., et al., Synthesis 1994,639-666; Griffith, w.; Ley, S. Aldrichimica Acta 1990, 23, 13-19) withNMO (N-methylmorpholine oxide) as the secondary oxidant (2.0 equiv)afforded the corresponding aldehydes 14 b and 14 c, respectively. Theseprocedures completed the preparation of the volatile and sensitivealdehydes which were used in the subsequent aldol condensations forinstalling the C15-C24/25 upper side chain of the myxopyronins.

Titanium (IV) Promoted Aldol Condensation and Completion of theSyntheses. The introduction of the upperside chains of myxopyronin A andB was carried out utilizing a Ti(IV) tetrachloride promoted aldolcondensation between the ethyl ketone of 15 a and aldehydes 14 b and 14c (FIG. 19). The titanium enolate was generated at −78° C. by treatmentof ethyl ketone 15 a with TiCl₄ (4.0 equiv) and DIPEA (4.8 equiv). Thederived enolate was condensed with freshly prepared aldehyde 14 b/c at−78° C. for 48-56 h to directly afford after in situ dehydration therespective (E,E)-dienones 17 c and 17 d. With both side chains installedonto the a-pyrone core, completion of the individual syntheses requiredthe conversion of the 60 ,β-unsaturated methyl ester to themethylcarbamate. This was initiated by a LiOH (10 equiv, THF/H₂O 4:1, 15h) promoted hydrolysis of the methylesters which afforded the freecarboxylic acids 17 c and 17 d in quantitative yield. The vinylcarbamate was introduced by a modified Curtius rearrangement (Overman,L., et al., J. Org. Chem. 1978, 43, 2164-2167) employingethylchloroformate and NaN₃. This sequence completed the assembly of thelower side and achieved the synthesis of (±)-myxopyronin A and B.

Biological Evaluation of (±)-Myxopyronin A & B. The biologicalactivities of the myxypyronins were evaluated with an in vitrotranscription assay using E. coli RNA polymerase (FIGS. 20a,b). Themyxopyronins A/B were isolated as a mixture of natural productscontaining a 9:1 ratio of A and B. The synthetic (±)-myxopyronin A (FIG.20a) is equally potent as the natural product mixtures. As a validationof the in vitro transcription assay, a known transcription inhibitor,rifampicin, was included in the assay. As a comparison, the syntheticmyxypyronin A and B were tested against E. coli RNA polymeraseseparately and myxopyrorin B is shown to be a more potent molecule thanA (FIG. 20b).

Table II summarizes in vitro IC₅₀ and MIC values obtained from thecell-based evaluation of the myxopyronins using both gram-positive andgram-negative bacteria. The data show that myxopyronins have in vivocell-based activities against rifampicin-resistant bacteria. Incomplement to the in vitro transcription activities, myxopyronin B isalso shown to have up to 30 fold more potent cell-based activities thanA (Table II).

TABLE II In vitro transcription assays (IC₅₀) and MIC values of thesynthetic myxopyronins and the mixture of natural products In vitrotranscription (E. coli MIC MIC MIC MIC Com- RNAP) (μg/mL) (μg/mL)(μg/mL) (μg/mL) pound IC₅₀ (μg/mL) E. coli E. coli* S. aureus S. aureus*(R)-Myxo 8 200 5 4 5 A/B (natural mixture) (±)-Myxo 5 >30 2 0.5 0.5 B(±)-Myxo 20 >30 4-8 15 8 A *Mutant strain that has permeabilized cellwall.

The present invention therefore provides a highly convergent syntheticpathways to myxopyronin A and B, as well as analogs and derivativesthereof. Biological evaluation of these agents against RNA polymerasefor a variety of bacteria including mammalian culture cells demonstratestheir considerable utility as antibacterial agents.

What is claimed is:
 1. A compound having the structure:

wherein R is C₁₋₉alkyl, and wherein R₁ is H, C₁₋₉alkyl, benzyl,optionally substituted phenyl, OH, NH₂, alklamine, dialkylamine, oroptionally substituted phenylamine.
 2. An antibacterial therapeuticcomposition comprising a compound of claim 1 and a pharmaceuticallyacceptable carrier.
 3. A process of preparing a myxopyronin having thestructure:

wherein R is C₁₋₉alkyl, and wherein R₁ is NH₂, alkylamine, dialkylamine,or optionally substituted phenylamine; which comprises: (a) saponifyinga pyrone ketone having the structure:

wherein R₀ is C₁₋₉alkyl; to form a pyrone acid; and (b) acylating thepyrone acid to form a pyrone anhydride; (c) reacting the pvroneanhydride formed in step (b) with an azide salt to form a pyrone acylazide; and (d) reacting the pyrone acyl azide formed in step (c) with anammonia, alkylamine, dialkylamine or optionally substituted phenvlamineto form the myxopyronin.
 4. The process of claim 3 wherein the pyroneketone is saponified in the presence of a hydroxide salt.
 5. The processof claim 4 wherein the hydroxide salt is LiOH, NaOH, KOH, ammoniumhydroxide, tetramethylammonium hydroxide, tetraethyl-ammonium hydroxide,tetra-n-propylammonium hydroxide or tetra-n-butylammonium hydroxide. 6.The process of claim 3 wherein the alkylamine is methylamine.
 7. Theprocess of claim 3 wherein the pyrone is treated with alkyl haloformate,and subsequently with an azide salt.
 8. The process of claim 7 whereinthe alkylhaloformate is methyl or ethyl chloroformate, and the azidesalt is LiN₃ or NaN₃.
 9. The process of claim 3 wherein R is methyl. 10.The process of claim 3 wherein R is ethyl.